Staphylococcal immunotherapeutics via donor selection and donor stimulation

ABSTRACT

A method and composition for the passive immunization of patients infected with or susceptible to infection from  Staphylococcus  bacteria such as  S. aureus  and  S. epidermidis  infection is provided that includes the selection or preparation of a donor plasma pool with high antibody titers to carefully selected  Staphylococcus  adhesins or MSCRAMMs, or fragments or components thereof, or sequences with substantial homology thereto. The donor plasma pool can be prepared by combining individual blood or blood component samples which have higher than normal titers of antibodies to one or more of the selected adhesins or other proteins that bind to extracellular matrix proteins, or by administering carefully selected proteins or peptides to a host to induce the expression of desired antibodies, and subsequently recovering the enhanced high titer serum or plasma pool from the treated host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/091,494, filed Mar. 7, 2002, which was a divisional application ofU.S. Ser. No. 09/386,960, filed Aug. 31, 1999, now U.S. Pat. No.6,692,739, which claims the benefit of provisional application U.S. Ser.No. 60/098,449, filed Aug. 31, 1998.

FIELD OF THE INVENTION

The invention is in the field of biological products for the treatment,prevention and diagnosis of bacterial infections.

BACKGROUND OF THE INVENTION

The staphylococci are Gram-positive spherical cells, usually arranged ingrape-like irregular clusters. Some are members of the normal flora ofthe skin and mucous membranes of humans, others cause suppuration,abscess formation, a variety of pyogenic infections, and even fatalsepticemia. Pathogenic staphylococci often hemolyze blood, coagulateplasma, and produce a variety of extracellular enzymes and toxins. Themost common type of food poisoning is caused by a heat-stablestaphylococci enterotoxin.

The genus Staphylococcus has at least 30 species. Three main species ofclinical importance are Staphylococcus aureus, Staphylococcusepidermidis, and Staphylococcus haemolyticus. Staphylococcus aureus iscoagulase-positive, which differentiates it from the other species. S.aureus is a major pathogen for humans. Almost every person has some typeof S. aureus infection during a lifetime, ranging in severity from foodpoisoning or minor skin infections to severe life-threateninginfections. The coagulase-negative staphylococci are normal human florawhich sometimes cause infection, often associated with implanteddevices, especially in very young, old and immunocompromised patients.Approximately 75% of the infections caused by coagulase-negativestaphylococci are due to parasitic S. epidermidis. Infections due toStaphylococcus haemolyticus, Staphylococcus hominis, and other speciesare less common. S. saprophyticus is a relatively common cause ofurinary tract infections in young women.

Staphylococcus bacteria such as S. aureus thus cause a spectrum ofinfections that range from cutaneous lesions such as wound infections,impetigo, and furuncles to life-threatening conditions that includepneumonia, septic arthritis, sepsis, endocarditis, and biomaterialrelated infections. S. aureus colonization of the articular cartilage,of which collagen is a major component, within the joint space appearsto be an important factor contributing to the development of septicarthritis. Hematogenously acquired bacterial arthritis remains a seriousmedical problem. This rapidly progressive and highly destructive jointdisease is difficult to eradicate. Typically less than 50% of theinfected patients failing to recover without serious joint damage. S.aureus is the predominant pathogen isolated from adult patients withhematogenous and secondary osteomyelitis.

In hospitalized patients, Staphylococcus aureus is a major cause ofinfection. Initial localized infections of wounds or indwelling medicaldevices can lead to more serious invasive infections such as septicemia,osteomyelitis, mastitis and endocarditis. In infections associated withmedical devices, plastic and metal surfaces become coated with hostplasma and matrix proteins such as fibrinogen and fibronectin shortlyafter implantation. The ability of Staphylococcus bacteria such as S.aureus to adhere to these proteins is essential to the initiation ofinfection. Vascular grafts, intravenous catheters, artificial heartvalves, and cardiac assist devices are thrombogenic and prone tobacterial colonization. S. aureus is the most damaging pathogen of suchinfections, and other Staphylococci bacteria such as S. epidermidis arealso responsible for a significant amount of dangerous infections,particularly those associated with implanted devices.

There is a strong and rapidly growing need for therapeutics to treatinfections from Staphylococcus bacteria such as S. aureus and S.epidermidis infections which are effective against antibiotic resistantstrains of the bacteria. The U.S. National Institutes for Health hasrecently indicated that this goal is now a national priority.

MSCRAMMs

The successful colonization of the host is a process required for mostmicroorganisms to cause infections in animals and humans. Microbialadhesion is the first crucial step in a series of events that caneventually lead to disease. Pathogenic microorganisms colonize the hostby attaching to host tissues or serum conditioned implantedbiomaterials, such as catheters, artificial joints, and vascular grafts,through specific adhesins present on the surface of the bacteria.MSCRAMMs (Microbial Surface Components Recognizing Adhesive MatrixMolecules) are a family of cell surface adhesins that recognize andspecifically bind to distinct components in the host's extracellularmatrix. Once the bacteria have successfully adhered to and colonizedhost tissues, their physiology is dramatically altered and damagingcomponents such as toxins and proteolytic enzymes are secreted.Moreover, adherent bacteria often produce a biofilm and quickly becomemore resistant to the killing effect of most antibiotics.

For example, S. aureus is known to express a repertoire of differentMSCRAMMs that can act individually or in concert to facilitate microbialadhesion to specific host tissue components. MSCRAMMs provide anexcellent target for immunological attack by antibodies. The presence ofthe appropriate anti-MSCRAMM high affinity antibodies has a double-edgedattack, first the antibodies prevent microbial adherence and second theincreased titers of MSCRAMM antibodies facilitate a rapid clearance ofthe organism from the body through bacterial lysis, opsonization,phagocytosis and complement activation.

Passive Immunization to Bacterial Infections

Immunoglobulins (A, D, E, G, and M) are used by the body as a primarydefense to infections. Complement, available as a precursor proteinwhich is activated by the presence of microorganisms and globulins, alsoexhibits antibacterial activities. After previous antigenic exposure,the immune system produces a series of globulins which attach to andcoat bacteria or neutralize viruses so that they are readily recognized,phagocytized and destroyed by neutrophils and macrophages. Foreignproteins of invading organisms also stimulate a humoral immune responsewhich over a period of time from three to six weeks amplifies the numberof cells designed to recognize and destroy specific invaders.

In the last decade, intravenous immunoglobulin (IVIG) therapy has becomea major treatment regime for bacterial infections, especially inimmunocompromised patients (Siber, New Eng. J. Med., 327:269-271, 1992).IVIG therapy has exhibited efficacy against more than thirty-fivediseases caused by immunopathologic mechanisms. Passive immunizationagainst infections has been particularly successful with immuneglobulins specific for tetanus, hepatitis B, rabies, chicken pox andcytomegalovirus. There has been an inconsistent and disappointingresponse to the use of immunoglobulins to prevent nosocomial infections,likely due to the variety of strains of bacteria found in hospitals andthe emergence of new serotypes. Passive immunization requires thepresence of high and consistent titers of antibodies to the infectingpathogens.

Supplemental immunoglobulin therapy has been shown to provide somemeasure of protection against certain encapsulated bacteria such asHemophilus influenzae and Streptococcus pneumoniae. Infants who aredeficient in antibody are susceptible to infections from these bacteriaand bacteremia and sepsis are common. When anti-Streptococcal andanti-Hemophilus antibodies are present, they provide protection bypromoting clearance of the respective bacteria from the blood. In thecase of antibody to Staphylococcus, the potential use of supplementalimmunoglobulin to prevent or treat infection has been much less clear.

Early attempts to treat Staphylococcus infections focused on thepotential use of supplemental immunoglobulin to boost peritonealdefenses, such as opsonic activity, in patients receiving continuousambulatory peritoneal dialysis. Standard intravenous immunoglobulin(IVIG) was shown to have lot to lot variability for opsonic activity toS. epidermidis (L. A. Clark and C. S. F. Easmon, J. Clin. Pathol. 39:856(1986)). In this study, one third of the IVIG lots tested had pooropsonization with complement, and only two of fourteen were opsonicwithout complement. Thus, despite the fact that the IVIG lots were madefrom large plasma donor pools, good opsonic antibody to S. epidermidiswas not uniformly present. Moreover, this study did not examine whetherIVIG could be used to prevent or treat S. epidermidis infections orbacterial sepsis.

Prior studies have associated coagulase-negative Staphylococcusbacteria, such as S. epidermidis, as the most common species causingbacteremia in neonates receiving lipid emulsion infusion (Freeman, J. etal., N. Engl. J. Med. 323:301, 1990). These neonates had low levels ofopsonic antibody to S. epidermidis despite the fact that the sera hadclearly detectable levels of IgG antibodies to S. epidermidispeptidoglycan (Fleer, A. et al., J. Infect. Dis. 2:426, 1985). This wassurprising because anti-peptidoglycan antibodies were presumed to be theprincipal opsonic antibodies. Thus, while suggesting that neonatalsusceptibility to S. epidermidis might be related to impaired opsonicactivity, these studies also suggested that many antibodies directedagainst S. epidermidis are not opsonic and would not be capable ofproviding protection when given passively to neonates.

In addition, an antigen binding assay was used to analyze IgG antibodyto S. epidermidis in patients with uncomplicated bacteremia and thosewith bacteremia and endocarditis (F. Espersen et al., Arch. Intern. Med.147:689 (1987)). This assay used an ultrasonic extract of S. epidermidisto identify S. epidermidis specific IgG. None of the patients withuncomplicated bacteremia had IgG antibodies to S. epidermidis. Thesedata suggest that IgG does not provide effective eradication of S.epidermidis from the blood. In addition, 89% of bacteremic patients withendocarditis developed high levels of IgG to S. epidermidis. In thesepatients, IgG was not protective since high levels of IgG antibody wereassociated with serious bacteremia and endocarditis. Based on thesestudies, the protective role of IgG in S. epidermidis sepsis andendocarditis was questionable, especially in the presence of immaturity,debilitation, intralipid infusion, or immunosuppression.

Animal studies in the literature that demonstrated immunoglobulinprotection against Staphylococcus infections have shown strainspecificity by enzyme-linked immunosorbent assays (ELISA) and haveutilized normal adult mice in protection studies. Animal modelstypically have used mature animals with normal immunity with unusuallyvirulent strains or overwhelming-challenge doses of bacteria. Humanpatients are generally immunologically immature or debilitated. Humanpatients also get somewhat indolent infections with low virulencepathogens such as S. epidermidis with death usually attributable tosecondary complications. Models that have used unusual strains oroverwhelming bacterial doses, generally induce rapid fulminant death.These are important factors since antibodies generally work in concertwith the host cellular immune system (neutrophils, monocytes,macrophages and fixed reticuloendothelial system). The effectiveness ofantibody therapy may therefore be dependent on the functionalimmunologic capabilities of the host. To be predictive, animal modelsmust closely emulate the clinical condition in which the infection wouldoccur and capture the setting for therapy. Moreover, the animal studieshave yielded inconsistent results.

One model has been reported which used an unusually virulent strain ofS. epidermidis. Infected-mature mice developed 90 to 100% mortalitywithin 24 to 48 hours (K. Yoshida et al., Japan. J. Microbiol. 20:209(1976)). Antibody to S. epidermidis surface polysaccharide wasprotective in these mice. Protection was shown to occur with an IgMfraction, but not the IgG fraction (K. Yoshida and Y. Ichiman, J. Med.Microbiol. 11:371 (1977)). This model, however, presents a pathologywhich is very different from that seen in typically infected patients.Intraperitoneally-challenged mice developed symptoms of sepsis withinminutes of receiving the injection and died in 24 to 48 hours. Thisparticular pathology is not observed in Staphylococcus infected humans.The highly virulent strain of S. epidermidis may represent an atypicaltype of infection. moreover, isolates of S. epidermidis from infectedhumans did not kill mice in this model.

In 1987, these animal studies were extended to include the evaluation ofantibodies in human serum against selected virulent strains of S.epidermidis (Y. Ichiman et al., J. Appl. Bacteriol. 63:165 (1987)). Incontrast to the previous data, protective antibody was found in the IgA,IgM and IgG immunoglobulin fractions. A definitive role for any singleclass of immunoglobulin (IgG, IgM, IgA) could not be established.

In this animal model, normal adult mice were used and mortality wasdetermined. Death was considered to be related to the effect of specificbacterial toxins, not sepsis (K. Yoshida et al., Japan J. Microbiol.20:209 (1976)). Most clinical isolates did not cause lethal infections,and quantitative blood cultures were not done. Moreover, this studyprovided little insight as to whether antibody could successfullyprevent or treat S. epidermidis sepsis in immature or immunosuppressedpatients.

In a later study, serotype specific antibodies directed against S.epidermidis capsular polysaccharides were tested in the animal model.Results showed that serotype-specific antibodies were protective, butthat each antibody was directed against one serotype as measured byELISA. Protection was equally serotype specific. Protection againstheterologous strains did not occur. In addition, it was concluded thatprotection was afforded by the IgM antibody.

There has been no compelling evidence that IVIG would be effective totreat S. epidermidis infections or sepsis, particularly where thepatients are immature or immune suppressed or where multiple S.epidermidis serotypes are involved. Thus, for example, a recent andextensive review of the pathogenesis, diagnosis, and treatment of S.epidermidis infections does not include immunoglobulin as a potentialprophylactic or therapeutic agent (C. C. Patrick, J. Pediatr. 116:497(1990)). In addition, there have been no U.S. patents which describe theeffective use of IVIG therapy in conjunction with antibodies to MSCRAMMssuch as described above.

U.S. Pat. No. 5,505,945 discloses compositions for passive immunity thatcontain a full repertoire of immunoglobulins, including IgA, IgM, andIgG to combat infections from microorganisms and viruses at wound,surgical, or burn sites. The compositions contain elevated antibodytiters for several pathogens, including S. aureus, Coagulase NegativeStaphylococci Enterococci, S. epidermidis, P. aeruginose, E. coli, andEnterobacter spp. However, these compositions are specifically designedto avoid the use of intravenous immunoglobulin or IVIG therapy, andinstead are applied in the form of ointments, creams, sprays and thelike which are designed for topical application only.

U.S. Pat. No. 4,717,766 discloses a method of preparing high titeranti-respiratory syncytial virus intravenous immunoglobulins.

U.S. Pat. No. 5,219,578 describes a composition and method forimmunostimulation in mammals, and specifically describes the isolationof an IgG fraction from goats free from foreign or artificially inducedantigens and the utilization of the isolated immunoglobulins fraction toinduce a stimulated immune response.

U.S. Pat. No. 5,548,066 describes a method for drawing blood from adonor animal, permitting blood to clot, separating liquid from cellularmaterial, and then clarifying, concentrating and sterilizing theproduct.

U.S. Pat. No. 4,412,990 discloses an intravenous pharmaceuticalcomposition containing immunoglobulin (IgG) and fibronectin thatexhibits a synergistic opsonic activity which results in enhancedphagocytosis of bacteria, immune complexes and viruses.

U.S. Pat. No. 4,994,269 discloses the topical use of monoclonalantibodies for the prevention and treatment of experimental P.aeriginosa lung infections. Specifically, the antibodies areadministered via aerosol spray to the lungs. Results show beneficialeffects in the treatment of affected patients.

U.S. Pat. No. 4,714,612 discloses the use of a non-specific gammaglobulin IgG in a mouthwash for the prevention of gingivitis. Anothermouthwash with monoclonal antibodies is described by Ma et al. in Arch.Oral Biol., 35 Supp: 115S-122S, in 1990. The monoclonal antibodies werespecific for Streptococcus mutans, and patients treated with themouthwash remained free of S. mutans for up to two years. Those who didnot take the mouthwash experiences recolonization of S. mutans withintwo days.

U.S. Pat. Nos. 5,718,889 and 5,505,945 describe the direct, concentratedlocal delivery of passive immunity which is accomplished by applying acomposition having a full repertoire of immunoglobulins (IgG, IgM andIgA) to biomaterials, implants, tissues, and wound and burn sites.

U.S. Pat. No. 5,571,511 describes the use of immunoglobulin fromindividual samples or pools of serum, plasma, whole blood, or tissue forthe treatment of a Staphylococcus infection. Immunoglobulin isidentified by performing a first assay to identify immunoglobulin whichis reactive with a preparation of a first Staphylococcus organism,performing a second assay to identify immunoglobulin which is reactivewith a preparation of a second Staphylococcus organism, and selectingimmunoglobulin which is reactive with the preparations from both thefirst and second Staphylococcus organisms. Reactivity is determined inimmunological assays which may be binding assays, opsonization assays,or clearance assays. Preferably, the preparations of the first and thesecond Staphylococcus organisms are derived from different serotypes ordifferent species, such as S. epidermidis and S. aureus, and morepreferably, the first preparation is from S. epidermidis (Hay, ATCC55133).

U.S. Pat. No. 5,412,077 describes the screening of plasma samples foreffective antibody titers for the treatment or prophylaxis of aninfection caused by a respiratory virus.

Accordingly, there still remains a need to provide more effectiveproducts and methods which make use of antibodies against MSCRAMMs andcan be utilized in methods of intravenous immunoglobulin therapy so asto prevent and/or treat Staphylococcus infections, and preferably thosethat can exhibit a broad spectrum immunization against various strainsof Staphylococcus bacteria.

Active Immunization to Bacterial Infections

Historically, studies on bacterial adherence have focused primarily onGram-negative bacteria, which express a wide variety of adhesiveproteins on their cell surface (Falkow, S., et al., Cell, 65:1099-1102,1992). These adhesins recognize specific glycoconjugates exposed on thesurface of host cells (particularly epithelial layers). Employing thelectin-like structures in attachment allows the microorganism toefficiently colonize the epithelial surfaces. This provides the bacteriaan excellent location for replication and also the opportunity todisseminate to neighboring host tissues. It has been demonstrated thatimmunization with pilus adhesins can elicit protection against microbialchallenge, such as in Hemophilus influenza induced otitis media in achinchilla model (Sirakova et al., Infect. Immun, 62(5):2002-2020,1994), Moraxella bovis in experimentally induced infectious bovinekeratoconjunctivitis (Lepper et al., Vet Microbiol, 45(2-3):129-138,1995), and E. coli induced diarrhea in rabbits (McQueen et al., Vaccine,11:201-206, 1993). In most cases, immunization with adhesins leads tothe production of immune antibodies that prevent infection by inhibitingbacterial attachment and colonization, as well as enhancing bacterialopsonophagocytosis and antibody-dependent complement-mediated killing.

The use of molecules that mediate the adhesion of pathogenic microbes tohost tissue components as vaccine components is emerging as a criticalstep in the development of future vaccines. Because bacterial adherenceis the critical first step in the development of most infections, it isan attractive target for the development of novel vaccines. An increasedunderstanding of the interactions between MSCRAMMs and host tissuecomponents at the molecular level coupled with new techniques inrecombinant DNA technology have laid the foundation for a new generationof subunit vaccines. Entire or specific domains of MSCRAMMs, either intheir native or site-specifically altered forms, can now be produced.Moreover, the ability to mix and match MSCRAMMs from differentmicroorganisms creates the possibility of designing a single vaccinethat will protect against multiple bacteria.

The recent clinical trials with a new subunit vaccine against whoopingcough, consisting of the purified Bordatella pertussis MSCRAMMsfilamentous hemagglutinin and pertactin, in addition to an inactivatedpertussis toxin, are a prime example of the success of this type ofapproach. Several versions of the new acellular vaccine were shown to besafe and more efficacious than the old vaccine that contained wholebacterial cells (Greco et al., N Eng J Med, 334:341-348, 1996;Gustaffson et al., N Eng J Med, 334:349-355, 1996).

Natural immunity to Staphylococcus infections remains poorly understood.Typically, healthy humans and animals exhibit a high degree of innateresistance to Staphylococcus bacteria such as S. aureus. Protection isattributed to intact epithelial and mucosal barriers and normal cellularand humoral responses. Titers of antibodies to S. aureus components areelevated after severe infections (Ryding et al., J. Med Microbiol,43(5):328-334, 1995), however to date there is no serological evidenceof a correlation between antibody titers and human immunity.

Over the past several decades live, heat-killed, and formalin fixedpreparations of S. aureus cells have been tested as vaccines to preventstaphylococcal infections. A multicenter clinical trial was designed tostudy the effects of a commercial vaccine, consisting of astaphylococcus toxoid and whole killed staphylococci, on the incidenceof peritonitis, exit site infection, and S. aureus nasal carriage amongcontinuous peritoneal dialysis patients (Poole-Warren, L. A., et al.,Clin Nephrol, 35:198-206, 1991). Although immunization with the vaccineelicited an increase in the level of specific antibodies to S. aureus,the incidence of peritonitis was unaffected. Similarly, immunization ofrabbits with whole cells of S. aureus could not prevent or modify anystage in the development of experimental endocarditis, reduce theincidence of renal abscess, or lower the bacterial load in infectedkidneys (Greenberg, D. P., et al., Infect Immun, 55:3030-3034, 1987).

Currently there is no FDA approved vaccine for the prevention of S.aureus infections. However, a S. aureus vaccine (StaphVAX), based on thecapsular polysaccharide, is currently being developed by NABI (NorthAmerican Biologicals Inc.). This vaccine consists of type 5 or type 8capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A(rEPA). The vaccine is designed to induce type-specific opsonicantibodies and enhance opsonophagocytosis (Karakawa, W. W., et al.,Infect Immun, 56:1090-1095, 1988). Using a refined lethal challengemouse model (Fattom, A., et al., Infect Immun, 61:1023-1032, 1996) ithas been shown that intraperitoneal infusion of type 5 specific IgGreduces the mortality of mice inoculated intraperitoneally with S.aureus. The type 5 capsular polysaccharide-rEPA vaccine has also beenused to vaccinate seventeen patients with end-stage renal disease(Welch, et al., J Amer Soc Nephrol, 7(2):247-253, 1996). Geometric mean(GM) IgG antibody levels to the type 5 conjugate increased between 13and 17-fold after the first immunization, however no additionalincreases could be detected after additional injections. Moreover, thesevaccination regimens were not able to treat a variety of bacterialstrains.

Interestingly, the GM IgM levels of the vaccinated patients weresignificantly lower than control individuals. Supported by the animalstudies, the vaccine has recently completed a Phase II trial incontinuous ambulatory peritoneal dialysis patients. The clinical trialshowed the vaccine to be safe but ineffective in preventingstaphylococcal infections (NABI SEC FORM 10-K405, 12/31/95). Twopossible explanations for the inability of StaphVAX to preventinfections related to peritoneal dialysis in vaccinated patients arethat the immunogenicity of the vaccine was too low due to suboptimalvaccine dosing or that antibodies in the bloodstream are unable toaffect infection in certain anatomic areas, such as the peritoneum.

Incidence of gram-positive bacteria related sepsis is increasing. Infact between one-third and one-half of all cases of sepsis are caused bygram-positive bacteria, particularly S. aureus and S. epidermidis. Inthe United States, it can be estimated that over 200,000 patients willdevelop gram-positive related sepsis this year. Using a mouse model(Bremell, et al., Infect Immun, 59(8):2615-2623, 1991), it has beenclearly demonstrated in PCT WO 97/43314 that active immunization withM55 domain of the Col-binding MSCRAMM can protect mice against sepsisinduced death. Mice were immunized subcutaneously with either M55 or acontrol antigen (bovine serum albumin) and then challenged intravenouslywith S. aureus. Eighty-three percent (35/42) of the mice immunized withM55 survived compared to only 27% of the BSA immunized mice (12/45).This a compilation of three separate studies.

Schennings et al. demonstrated that immunization with fibronectinbinding protein from S. aureus protects against experimentalendocarditis in rats (Micro Pathog, 15:227-236, 1993). Rats wereimmunized with a fusion protein (gal-FnBP) encompassingbeta-galactosidase and the domains of fibronectin binding protein fromS. aureus responsible for binding to fibronectin. Antibodies againstgal-FnBP were shown to block the binding of S. aureus to immobilizedfibronectin in vitro. Endocarditis in immunized and non-immunizedcontrol rats was induced by catheterization via the right carotidartery, resulting in damaged aortic heart valves which became covered byfibrinogen and fibronectin. The catheterized rats were then infectedintravenously with 1×10 5 cells of S. aureus. The number of bacteriaassociated with aortic valves was determined 1½ days after the challengeinfection and a significant difference in bacterial numbers betweenimmunized and non-immunized groups was then observed.

A mouse mastitis model was used by Mamo, et al., in 1994 (Vaccine,12:988-992) to study the effect of vaccination with fibrinogen bindingproteins (especially FnBP-A) and collagen binding protein from S. aureusagainst challenge infection with S. aureus. The mice vaccinated withfibrinogen binding proteins showed reduced rates of mastitis comparedwith controls. Gross examination of challenged mammary glands of miceshowed that the glands of mice immunized with fibrinogen bindingproteins developed mild intramammary infection or had no pathologicalchanges compared with glands from control mice. A significantly reducednumber of bacteria could be recovered in the glands from mice immunizedwith fibrinogen binding proteins as compared with controls. Mamo thenfound that vaccination with FnBP-A combined with staphylococcal alphatoxoid did not improve the protection (Mamo, et al., Vaccine,12:988-992, 1994). Next, Mamo, et al., immunized mice with only collagenbinding protein, which did not induce protection against the challengeinfection with S. aureus.

Whole killed staphylococci were included in a vaccine study in humansundergoing peritoneal dialysis (Poole-Warren, et al., ClinicalNephrology 35:198-206, 1991). In this clinical trial, a commerciallyavailable vaccine of alpha-hemolysin toxoid combined with a suspensionof whole killed bacteria) was administered intramuscularly ten timesover 12 months, with control patients receiving saline injections.Vaccination elicited significant increases in the levels of antibodiesto S. aureus cells in the peritoneal fluid and to alpha-hemolysin in theserum. However, immunization did not reduce the incidences ofperitonitis, catheter-related infections or nasal colonization amongvaccine recipients. The lack of protective efficacy in this trial wasattributed to a suboptimal vaccine formulation.

Secreted proteins have been explored as components of subcellularvaccines. The alpha toxin is among the most potent staphylococcalexotoxins; it has cytolytic activity, induces tissue necrosis and killslaboratory animals. Immunization with formaldehyde-detoxified alphatoxin does not protect animals from systemic or localized infections,although it may reduce the clinical severity of the infections (Ekstedt,R. D., The Staphylococci, 385-418, 1972)

One study has evaluated the protective efficacy of antibodies to the S.aureus microcapsule in an experimental model of staphylococcal infection(Nemeth, J. and Lee, J. C., Infect. Immun., 61:1023-1032, 1993). Ratswere actively immunized with killed, microencapsulated bacteria orpassively immunized with high-titer rabbit antiserum specific for thecapsular polysaccharide. Control animals were injected with saline orpassively immunized with normal rabbit serum. Protection againstcatheter-induced endocarditis resulting from intravenous challenge withthe same strain was then evaluated. Despite having elevated levels ofanticapsular antibodies, the immunized animals were susceptible tostaphylococcal endocarditis and immunized and control animals hadsimilar numbers of bacteria in the blood.

As described in the Detailed Description of the Invention hereinbelow, anumber of patents and published patent applications describe the genesequences for fibronectin, fibrinogen, collagen, elastin, and MHC IIantigen type binding proteins. These patents and patent applications areincorporated by reference in their entirety. These documents teach thatthe proteins, fragments, or antibodies immunoreactive with thoseproteins or fragments can be used in vaccinations for the treatment ofS. aureus infections. PCT/US97/087210 discloses the vaccination of micewith a combination of a collagen binding protein (M55 fragment), afibronectin binding peptide (formalin treated FnBPA (D1-D3)) and afibrinogen binding peptide (ClfA).

Despite the advances in the art of compositions for the treatment ofinfections from Staphylococcus bacteria such as S. aureus, there remainsa need to provide a more effective product, and preferably one thatexhibits a broad spectrum immunization against a variety ofStaphylococcus bacterial strains. As described in the DetailedDescription of the Invention, one approach to generating a prophylacticimmunotherapeutic against bacteria is to stimulate donors with a vaccinecontaining a combination of MSCRAMMs. This approach of generatinghyperimmune globulins can create a steady supply of plasma with highlevels of the specific types of disease fighting antibodies. MSCRAMMhyperimmune globulins can be used to provide passive immunity againstinfection in neonates, trauma patients, immunocompromised patients orpatients who are immediately at risk and do not have time to mount theirown antibody response. Hyperimmune globulins have a high benefit-to-costratio, can be produced from a nonhuman or human source and have a highlevel of physician acceptance based on past usage.

Therefore, it is an object of the invention to provide new therapeuticcompositions for active and passive immunization against Staphylococcusinfections.

It is another object of the present invention to provide active andpassive immunization against mastitis, arthritis, endocarditis,septicemia, osteomyelitis, furunculosis, cellulitis, pyemia, pneumonia,pyoderma, suppuration of wounds, food poisoning, bladder infections andother infectious diseases.

It is another object of the present invention to provide a therapeuticcomposition that immunizes against Staphylococcus bacteria such as S.aureus and S. epidermidis, increases the rate of opsonization andphagocytosis of a variety of Staphylococcus infections, and inducesenhanced intracellular killing of Staphylococcus bacteria.

It is another object of the present invention to provide animmunological serum against staphylococci.

It is another object of the present invention to provide such a serumwhich yields humoral and cellular immunity against staphylococci.

It is another object of the present invention to provide such a serumwhich imparts short-term immunity against staphylococci.

It is a further object of the present invention to provide methods fordetecting, diagnosing, treating, preventing or monitoring the progressof therapy for staphylococcal infections.

SUMMARY OF THE INVENTION

A method and composition for the passive immunization of patientsinfected with or susceptible to infection from Staphylococcus bacteriasuch as S. aureus and S. epidermidis infection is provided that includesthe selection or preparation of a donor plasma pool with high antibodytiters to carefully selected Staphylococcus adhesins or MSCRAMMs, orfragments or components thereof, or sequences with substantial homologythereto; purification, concentration, and treatment of the donor plasmapool as necessary to obtain immunoglobulin in a purified state that hasa higher than normal antibody titer to the selected adhesins; and thenadministration of an effective amount of the purified immunoglobulin tothe patient in need thereof. The donor plasma pool can be prepared, forexample, by combining individual blood or blood component samples whichhave higher than normal titers of antibodies to one or more of theselected adhesins or other proteins that bind to extracellular matrixproteins, or fragments or sequences with substantial homology thereto,to produce the desired composite. Kits for the identification of donorplasma pools with high titers of the selected adhesins are alsoprovided. In an alternative embodiment, a method for obtaining a donorplasma pool that is highly effective against Staphylococcus bacterialinfection is provided that includes administering carefully selectedproteins or peptides to a host to induce the expression of desiredantibodies, recovering the enhanced high titer serum or plasma pool fromthe host, optionally purifying and concentrating the immunoglobulin, andproviding it to a patient in need thereof.

A “high titer” of antibody in this context means the presence of anantibody which is immunoreactive with the selected adhesin or fragmentthereof which is 2-fold or greater, e.g., up to 10-20 more times higherthan that found in a normal population of 100 random samples of blood orblood components.

In one embodiment of the invention, a donor plasma composition isselected or prepared that has a high titer of antibodies to at least afibrinogen binding protein, such as Clumping factor A (“ClfA”) orClumping factor B (“ClfB”), or fragments or components thereof, or aprotein or fragment with sufficiently high homology thereto.

In another embodiment of the invention, a donor plasma composition isselected or prepared that has a high titer of antibodies to at least acollagen binding protein or peptide (or an appropriate site directedmutated sequence thereof), a fragment or component thereof, such as thecollagen binding domain protein M55, or a protein or fragment withsufficiently high homology thereto.

In another embodiment of the invention, a donor plasma composition isselected or prepared that has a high titer of antibodies to at least afibronectin binding protein or peptide (or an appropriate site directedmutated sequence thereof), or a protein or fragment with sufficientlyhigh homology thereto, as well as the fibrinogen binding protein A and B(ClfA or ClfB), or useful fragments or components thereof or a proteinor fragment with sufficiently high homology thereto.

In a further embodiment, a donor pool is selected or prepared that has ahigh titer of antibodies to at least the fibrinogen binding protein A(ClfA) and the fibrinogen binding protein B (ClfB), or useful fragmentsthereof or a protein or fragment with sufficiently high homologythereto.

In a still further embodiment, a donor pool is selected or prepared witha high titer of antibodies to at least a fibronectin binding protein orpeptide (or an appropriate site directed mutated sequence thereof), or aprotein or fragment with sufficiently high homology thereto, incombination with (I) high titer antibodies to the fibrinogen bindingprotein A and B (ClfA and ClfB), or a useful fragment thereof or aprotein or fragment with sufficiently high homology thereto; and (ii)high titer antibodies to a collagen binding protein or useful fragmentthereof.

In another embodiment, a donor pool is selected or prepared that has ahigh titer of antibodies as in any of the previous embodiments incombination with a high titer of antibodies to an elastin bindingprotein or peptide or a protein or fragment with sufficiently highhomology thereto.

In another embodiment, a donor pool is selected or prepared that has ahigh titer of antibodies as set forth in the embodiments above incombination with high titers of antibodies to a MHC II analogous proteinor peptide or a protein or fragment with sufficiently high homologythereto.

In an additional embodiment, a donor pool is selected or prepared thathas a high titer of antibodies to any of the embodiments above incombination with high titer of antibodies to one or more fibrinogenbinding proteins SdrC, SdrD or SdrE, or useful fragments thereof orproteins or fragments with sufficiently high homology thereto.

In still another embodiment, a donor pool is selected or prepared thathas a high titer of antibodies to at least the fibrinogen bindingprotein SdrC, the fibrinogen binding protein SdrD and the fibrinogenbinding protein SdrE or useful fragments thereof or a protein orfragment with sufficiently high homology thereto.

Kits are also provided that identify plasma pools with high titers ofthe desired antibodies. In one embodiment, a suitable amount ofantibodies to antibodies of the combination of proteins or peptides asdescribed herein can be immobilized on a solid support and arepreferably labeled with a detectable agent. Antibodies can beimmobilized to a variety of solid substrates by known methods. Suitablesolid support substrates include materials having a membrane or coatingsupported by or attached to sticks, beads, cups, flat packs, or othersolid support. Other solid substrates include cell culture plates, ELISAplates, tubes, and polymeric membranes. The antibodies can be labeledwith a detectable agent such as a fluorochrome, a radioactive label,biotin, or another enzyme, such as horseradish peroxidase, alkalinephosphatase and 2-galactosidase. If the detectable agent is an enzyme, ameans for detecting the detectable agent can be supplied with the kit. Apreferred means for detecting a detectable agent employs an enzyme as adetectable agent and an enzyme substrate that changes color upon contactwith the enzyme. The kit can also contain a means to evaluate theproduct of the assay, for example, a color chart, or numerical referencechart.

Preferably, the isolated immunoglobulin is of the IgG fraction orisotype, but isolated immunoglobulin is not restricted to any particularfraction or isotype and may be IgG, IgM, IgA, IgD, IgE, or anycombination thereof. It is also preferable that the isolatedimmunoglobulin be purely or antigenically human immunoglobulin, whichmay be obtained from human sources or made directly by the fusion ofhuman antibody producing cells with human antibody producing cells or bythe substitution of human DNA sequences for some of the nonhuman DNAsequences which code for the antibody while retaining the antigenbinding ability of the original antibody molecule.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a schematic representation of the peptides used inillustrative vaccine, MSCRAMM IV. This drawing illustrates the essentialfeatures of the collagen binding MSCRAMM CNA, fibrinogen binding MSCRAMMClfA, fibrinogen binding MSCRAMM ClfB and fibronectin binding MSCRAMMFnBPA proteins.

FIG. 2 is a time course graph of the immune response in MCSCRAMMvaccinated Rhesus Monkeys as shown by changes in antibody titers againstthe MSCRAMMs CNA, ClfA, ClfB and FnBPA, respectively. The titers wereanalyzed by ELISA and measured as changes in absorbance (quantified at405 nm) during each week over the course of a six-month period oftreatment following the original immunization with the antigen.

DETAILED DESCRIPTION OF THE INVENTION

A method and composition for the passive immunization of patientsinfected with or susceptible to Staphylococcus bacterial infection, suchas those caused by S. aureus or S. epidermidis, is provided thatincludes the selection or preparation of a donor plasma pool with highantibody titers to carefully selected Staphylococcus adhesins, orfragments thereof or sequences with substantial homology thereto;purification, concentration, and treatment of the donor plasma pool asnecessary to obtain immunoglobulin in a purified state that has a higherthan normal antibody titer to the selected staphylococcal adhesins; andthen administration of an effective amount of the purifiedimmunoglobulin to the patient in need thereof. The donor plasma pool canbe prepared, for example by, by combining individual blood samples whichhave higher than normal titers of antibodies to one or more of theselected adhesins or fragments or sequences with substantial homologythereto. Kits for the identification of donor plasma pools with hightiters of the selected adhesins are also provided. In an alternativeembodiment, a method for obtaining a donor plasma pool that is highlyeffective against Staphylococcus infection is provided that includesadministering carefully selected proteins or peptides to a host toinduce the expression of desired antibodies, recovering the enhancedhigh titer plasma pool from the host, optionally purifying andconcentrating the immunoglobulin, and providing it to a patient in needthereof.

Donor plasma pools are selected or prepared, purified, treated, and thenadministered in an effective amount to a patient in need thereof, whichinclude high titer antibodies to at least:

(i) a fibrinogen binding protein, such as Clumping factor A (“ClfA”) orClumping factor B (“ClfB”), or fragments or components thereof, or aprotein or fragment with sufficiently high homology thereto;

(ii) a collagen binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), a fragment or component thereof,such as the collagen binding domain protein M55, or a protein orfragment with sufficiently high homology thereto.

(iii) a fibronectin binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), or a protein or fragment withsufficiently high homology thereto, in combination with the fibrinogenbinding protein A and B (ClfA and ClfB), or useful fragments thereof ora protein or fragment with sufficiently high homology thereto;

(iv) the fibrinogen binding protein A (ClfA) and the fibrinogen bindingprotein B (ClfB), or useful fragments thereof or a protein or fragmentwith sufficiently high homology thereto;

(v) fibronectin binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), or a protein or fragment withsufficiently high homology thereto, in combination with (I) thefibrinogen binding protein A and B (ClfA and ClfB), or a useful fragmentthereof or a protein or fragment with sufficiently high homologythereto; and (ii) a collagen binding protein or useful fragment thereof;

(vi) components of any of the above in combination with an elastinbinding protein or peptide or a protein or fragment with sufficientlyhigh homology thereto;

(vii) components of any of the above embodiments in combination with aMHC II type binding protein or peptide or a protein or fragment withsufficiently high homology thereto;

(viii) components of any of the above embodiments in combination with athe fibrinogen binding proteins SdrC, SdrD or SdrE, or useful fragmentsthereof or proteins or fragments with sufficiently high homologythereto;

(ix) the fibrinogen binding protein SdrC, the fibrinogen binding proteinSdrD and the fibrinogen binding protein SdrE or useful fragments thereofor a protein or fragment with sufficiently high homology thereto; or

(x) proteins SdrF, SdrG and SdrH from coagulase-negative bacteria suchas S. epidermidis or useful fragments thereof or a proteins or fragmentswith sufficiently high homology thereto.

Isolated peptide fragments from wild-type or naturally occurringvariants and synthetic or recombinant peptides corresponding towild-type, naturally occurring variants or introduced mutations that donot correspond to a naturally occurring binding domain of a bindingprotein can be used to select or produce donor plasma pools.

I. Definitions

The terms FnBP-A protein, FnBP-B protein, ClfA protein, ClfB protein,SdrC protein, SdrD protein, SdrE protein, CNA protein, EbpS protein andMHCII protein are defined herein to include FnBP-A, FnBP-B, ClfA, ClfB,SdrC, SdrD, SdrE, CNA, EbpS and MHCII subdomains, respectively, andactive or antigenic fragments or components of FnBP-A, FnBP-B, ClfA,ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHCII proteins, respectively, orproteins or fragments having sufficiently high homology thereto. Activefragments or components of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE,CNA, EbpS and MHCII proteins are defined herein as peptides orpolypeptides capable of blocking the binding of staphylococci bacteriato extracellular matrix proteins of the host. Antigenic fragments ofFnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHCIIproteins are defined herein as peptides or polypeptides capable ofproducing an immunological response.

The term “adhesin” as used herein includes naturally occurring andsynthetic or recombinant proteins and peptides which can bind toextracellular matrix proteins and/or mediate adherence to host cells.

The term “amino acid” as used herein includes naturally occurring andsynthetic amino acids and includes, but is not limited to, alanine,valine, leucine, isoleucine, proline, phenylalanine, tryptophan,methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine,glutamate, aspartic acid, glutamic acid, lysine, arginine, andhistidine.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term as used herein includesmonoclonal antibodies, polyclonal, chimeric, single chain, bispecific,simianized, and humanized-antibodies as well as Fab fragments, includingthe products of an Fab immunoglobulin expression library.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

A “cell line” is a clone of a primary cell that is capable of stablegrowth in vitro for many generations.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thesequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, prokaryotic sequences, cDNAfrom eukaryotic mRNA, genetic DNA sequences from eukaryotic (e.g.,mammalian) DNA, and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

“DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g, restriction fragments),viruses, plasmids, and chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA

As used herein, the term “extracellular matrix proteins,” or ECM, refersto four general families of macromolecules—collagens, structuralglycoproteins, proteoglycans and elastins—that provide support andmodulate cellular behavior.

“Immunologically effective amounts” are those amounts capable ofstimulating a B cell and/or T cell response.

As used herein, the term “in vivo vaccine” refers to immunization ofanimals with proteins so as to elicit a humoral and cellular responsethat protects against later exposure to the pathogen.

The term “ligand” is used to include molecules, including those withinhost tissues, to which pathogenic bacteria attach.

The term “MHC II analogous proteins” as used herein refers tocell-surface molecules that are responsible for rapid graft rejectionsand are required for antigen presentation to T-cells.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an unacceptable allergic or similaruntoward reaction when administered to a human.

As used herein, a “protective antibody” is an antibody which confersprotection against infectious diseases caused by infection withstaphylococci, when used to passively immunize an naive animal.

As used herein, a “protective epitope” is an epitope which is recognizedby a protective antibody, and/or an epitope which, when used to immunizean animal, elicits an immune response sufficient to prevent or lessensthe severity for some period of time, of any one of the disorders whichcan result from infection with staphylococci.

The term “wound” is used herein to mean that normally coveringepithelial cellular layer, and other surface structures have beendamaged by mechanical, chemical or other influence.

By “immunologically effective amount” is meant an amount of a peptidecomposition that is capable of generating an immune response in therecipient animal. This includes both the generation of an antibodyresponse (B cell response), and/or the stimulation of a cytotoxic immuneresponse (T cell response). The generation of such an immune responsewill have utility in both the production of useful bioreagents, e.g.,CTLs and, more particularly, reactive antibodies, for use in diagnosticembodiments, and will also have utility in various prophylactic ortherapeutic embodiments.

II. Fibronectin-Binding MSCRAMMs

Fibronectin (Fn) is a 440-kDa glycoprotein found in the ECM and bodyfluids of animals. The primary biological function of fibronectinappears to be related to its ability to serve as a substrate for theadhesion of cells expressing the appropriate integrins. Severalbacterial species have been shown to bind fibronectin specifically andto adhere to a fibronectin-containing substratum. Most S. aureusisolates bind Fn, but do so in varying extents, which reflectsvariations in the number of MSCRAMM molecules expressed on the bacterialcell surface. The interaction between Fn and S. aureus is highlyspecific (Kuusela, P., Nature, 276:718-20, 1978). Fn binding is mediatedby two surface exposed proteins with molecular weights of 110 kDa, namedFnBP-A and FnBP-B. The primary Fn binding site consists of a motif of35-40 amino acids, repeated three to five times. The genes for thesehave been cloned and sequenced (Jonsson, K., et al., Eur. J. Biochem.,202:1041-1048, 1991). Potential applications for vaccination withanti-FnBP antibodies include, but are not limited to, bovine mastitis,endocarditis and wound infections.

WO-A-85/05553 discloses bacterial cell surface proteins havingfibronectin, fibrinogen, collagen, and or laminin binding ability.

U.S. Pat. Nos. 5,320,951 and 5,571,514 to Hook, et al., discloses thegene sequence of fibronectin binding protein A (fnbA), and biologicalproducts and methods based on this sequence.

U.S. Pat. No. 5,175,096 to Hook et al., discloses the gene sequence offnbB, a hybrid DNA molecule (fnbB) and biological products and methodsbased on this sequence.

U.S. Pat. No. 5,652,217 discloses an isolated and purified proteinhaving binding activity that is encoded by a hybrid DNA molecule from S.aureus of defined sequence.

U.S. Pat. No. 5,440,014 discloses a fibronectin binding peptide withinthe D3 homology unit of a fibronectin binding protein of S. aureus whichcan be used for vaccination of ruminants against mastitis caused bystaphylococcal infections, for treatment of wounds, for blocking proteinreceptors, for immunization of other animals, or for use in a diagnosticassay.

U.S. Pat. No. 5,189,015 discloses a method for the prophylactictreatment of the colonization of a S. aureus bacterial strain having theability to bind to fibronectin in a mammal that includes administeringto the mammal in need of treatment a prophylactically therapeuticallyactive amount of a protein having fibronectin binding properties, toprevent the generation of infections caused by a S. aureus bacterialstrain having the ability to bind fibronectin, wherein the protein has amolecular weight of 87 kDa to 165 kDa.

U.S. Pat. No. 5,416,021 discloses a fibronectin binding protein encodingDNA from Streptococcus dysgalactiae, along with a plasmid that includesDNA encoding for fibronectin binding protein from S. dysgalactiaecontained in E. coli, DNA encoding a fibronectin binding protein from S.dysgalactiae and an E. coli microorganism transformed by DNA encoding afibronectin binding protein from S. dysgalactiae.

It has been observed that antibodies to wild type fibronectin bindingprotein do not substantially inhibit the ability of S. aureus to bind tofibronectin, and thus do not exhibit a significant therapeutic effect invivo. PCT/US98/01222 discloses antibodies that block the binding offibronectin to fibronectin binding proteins. The antibodies were raisedagainst a site-directed mutated sequence of fibronectin binding proteinthat does not bind to fibronectin. It was identified that there is arapid complexing of fibronectin with fibronectin binding proteins andfragments in vivo. Peptide epitopes that do not bind to fibronectin,even though based on a fibronectin binding domain of a fibronectinbinding protein, do not form a complex with fibronectin in vivo. Thisallows antibodies to be made against the uncomplexed peptide epitope,which inhibit or block the binding of fibronectin to fibronectin bindingproteins.

III. Collagen-Binding MSCRAMMs

Collagen is the major constituent of cartilage. Collagen (Cn) bindingproteins are commonly expressed by staphylococcal strains. The Cnbinding MSCRAMM of S. aureus adheres to cartilage in a process thatconstitutes an important part of the pathogenic mechanism instaphylococcal infections. (Switalski, et al. Mol. Micro. 7(1), 99-107,1993) Cn binding by staphylococcal bacteria such as S. aureus is foundto play a role in at least, but not only, arthritis and septicemia. CNAproteins with molecular weights of 133, 110 and 87 kDa (Patti, J., etal., J. Biol. Chem., 267:4766-4772, 1992) have been identified. Strainsexpressing CNAs with different molecular weights do not differ in theirCn binding ability (Switalski, L. M., et al., Mol. Microbiol., 7:99-107,1993).

Staphylococcal strains recovered from the joints of patients diagnosedwith septic arthritis or osteomyelitis almost invariably express a CNA,whereas significantly fewer isolates obtained from wound infectionsexpress this adhesin. (Switalski, L. M., et al., Mol. Microbiol.,7:99-107, 1993) Similarly, S. aureus strains isolated from the bones ofpatients with osteomyelitis more often have an MSCRAMM recognizing thebone-specific protein, bone sialoprotein (BSP) (Ryden, C., et al,Lancet, 11:515-518, 1987). S. aureus colonization of the articularcartilage within the joint space appears to be an important factorcontributing to the development of septic arthritis.

The cloning, sequencing, and expression of a gene CNA, encoding a S.aureus CNA protein has been reported (Patti, J., et al., J. Biol. Chem.,267:4766-4772, 1992). The CNA gene encodes an 133-kDa adhesin thatcontains structural features characteristic of surface proteins isolatedfrom Gram-positive bacteria.

Recently, the ligand-binding site has been localized within theN-terminal half of the CNA (Patti, J. et al., Biochemistry,32:11428-11435, 1993). By analyzing the Col binding activity ofrecombinant proteins corresponding to different segments of the MSCRAMM,a 168-amino-acid long protein fragment (corresponding to amino acidresidues 151-318) that had appreciable Col binding activity wasidentified. Short truncations of this protein in the N or C terminusresulted in a loss of ligand binding activity but also resulted inconformational changes in the protein.

PCT WO 92/07002 discloses a hybrid DNA molecule which includes anucleotide sequence from S. aureus coding for a protein or polypeptidehaving collagen binding activity and a plasmid or phage comprising thenucleotide sequence. Also disclosed are an E. coli strain expressing thecollagen binding protein, a microorganism transformed by the recombinantDNA, the method for producing a collagen binding protein or polypeptide,and the protein sequence of the collagen binding protein or polypeptide.

Patti et al. (J of Biol Chem., 270, 12005-12011, 1995) disclose acollagen binding epitope in the S. aureus adhesin encoded by the CNAgene. In this study, the authors synthesized peptides derived from thesequence of the said protein and used them to produce antibodies. Someof these antibodies inhibit the binding of the protein to collagen.

PCT/US97/08210 discloses that certain identified epitopes of thecollagen binding protein (M55, M33, and M17) can be used to generateprotective antibodies. The application also discloses the crystalstructure of the CNA which provides critical information necessary foridentifying compositions which interfere with, or block completely, thebinding of Col to CNAs. The ligand-binding site in the S. aureus CNA anda 25-amino-acid peptide was characterized that directly inhibits thebinding of S. aureus to 125 I-labeled type II Col.

IV. Fibrinogen-Binding MSCRAMMs

Fibrin is the major component of blood clots, and fibrinogen/fibrin isone of the major host proteins deposited on implanted biomaterials.Considerable evidence exists to suggest that bacterial adherence tofibrinogen/fibrin is important in the initiation of device-relatedinfection. For example, as shown by Vaudaux et al., S. aureus adheres toin vitro plastic that has been coated with fibrinogen in adose-dependent manner (J. Infect. Dis. 160:865-875 (1989)). In addition,in a model that mimics a blood clot or damage to a heart valve, Herrmannet al. demonstrated that S. aureus binds avidly via a fibrinogen bridgeto platelets adhering to surfaces (J. Infect. Dis. 167: 312-322 (1993)).S. aureus can adhere directly to fibrinogen in blood clots formed invitro, and can adhere to cultured endothelial cells via fibrinogendeposited from plasma acting as a bridge (Moreillon et al., Infect.Immun. 63:4738-4743 (1995); Cheung et al., J. Clin. Invest. 87:2236-2245(1991)). As shown by Vaudaux et al. and Moreillon et al., mutantsdefective in the fibrinogen-binding protein clumping factor (ClfA)exhibit reduced adherence to fibrinogen in vitro, to explantedcatheters, to blood clots, and to damaged heart valves in the rat modelfor endocarditis (Vaudaux et al., Infect. Immun. 63:585-590 (1995);Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

An adhesin for fibrinogen, often referred to as “clumping factor,” islocated on the surface of S. aureus cells. The interaction betweenbacteria and fibrinogen in solution results in the instantaneousclumping of bacterial cells. The binding site on fibrinogen is locatedin the C-terminus of the gamma chain of the dimeric fibrinogenglycoprotein. The affinity is very high and clumping occurs in lowconcentrations of fibrinogen. Scientists have recently shown thatclumping factor also promotes adherence to solid phase fibrinogen, toblood clots, and to damaged heart valves (McDevitt et al., Mol.Microbiol. 11: 237-248 (1994); Vaudaux et al., Infect. Immun. 63:585-590(1995); Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

Two genes in S. aureus have been found that code for two Fg bindingproteins, ClfA and ClfB. The gene, clfA, was cloned and sequenced andfound to code for a polypeptide of 92 kDa. ClfA binds the gamma chain offibronectin, and ClfB binds the alpha and beta chains (Eidhin, et al.,Mol Micro, awaiting publication, 1998). ClfB is a cell wall associatedprotein with a predicted molecular weight of 88 kDa and an apparentmolecular weight of 124 kDa that binds both soluble and immobilizedfibrinogen and acts as a clumping factor.

The gene for a clumping factor protein, designated ClfA, has recentlybeen cloned, sequenced and analyzed in detail at the molecular level(McDevitt et al., Mol. Microbiol. 11: 237-248 (1994); McDevitt et al.,Mol. Microbiol. 16:895-907 (1995)). The predicted protein is composed of933 amino acids. A signal sequence of 39 residues occurs at theN-terminus followed by a 520 residue region (region A), which containsthe fibrinogen binding domain. A 308 residue region (region R), composedof 154 repeats of the dipeptide serine-aspartate, follows. The R regionsequence is encoded by the 18 basepair repeat GAY TCN GAY TCN GAY AGY inwhich Y equals pyrimidines and N equals any base. The C-terminus of ClfAhas features present in many surface proteins of gram-positive bacteriasuch as an LPDTG motif, which is responsible for anchoring the proteinto the cell wall, a membrane anchor, and positive charged residues atthe extreme C-terminus.

The platelet integrin alpha IIbβ3 recognizes the C-terminus of the gammachain of fibrinogen. This is a crucial event in the initiation of bloodclotting during coagulation. ClfA and alpha IIbβ3 appear to recognizeprecisely the same sites on fibrinogen gamma chain because ClfA canblock platelet aggregation, and a peptide corresponding to theC-terminus of the gamma chain (198-41 1) can block both the integrin andClfA interacting with fibrinogen (McDevitt et al., Eur. J. Biochem.247:416-424 (1997)). The fibrinogen binding site of alpha IIbβ33 isclose to, or overlaps, a Ca2+ binding determinant referred to as an “EFhand”. ClfA region A carries several EF hand-like motifs. Aconcentration of Ca2+ in the range of 3-5 mM blocks theseClfA-fibrinogen interactions and changes the secondary structure of theClfA protein. Mutations affecting the ClfA EF hand reduce or preventinteractions with fibrinogen. Ca2+ and the fibrinogen gamma chain seemto bind to the same, or to overlapping, sites in ClfA region A.

The alpha chain of the leukocyte integrin, alpha MB2, has an insertionof 200 amino acids (A or I domain) which is responsible for ligandbinding activities. A novel metal ion-dependent adhesion site (MIDAS)motif in the I domain is required for ligand binding. Among the ligandsrecognized is fibrinogen. The binding site on fibrinogen is in the gammachain (residues 190-202). It was recently reported that Candida albicanshas a surface protein, alpha Intip, having properties reminiscent ofeukaryotic integrins. The surface protein has amino acid sequencehomology with the 1 domain of Mβ2, including the MIDAS motif.Furthermore, Intlp binds to fibrinogen.

ClfA region A also exhibits some degree of sequence homology with alphaIntlp. Examination of the ClfA region A sequence has revealed apotential MIDAS motif. Mutations in putative cation coordinatingresidues in the D×S×S portion of the MIDAS motif in ClfA results in asignificant reduction in fibrinogen binding. A peptide corresponding tothe gamma-chain binding site for alpha Mβ2 (190-202) has been shown byO'Connell et al. to inhibit ClfA-fibrinogen interactions (O'Connell etal., J. Biol. Chem., in press). Thus it appears that ClfA can bind tothe gamma-chain of fibrinogen at two separate sites. The ligand bindingsites on ClfA are similar to those employed by eukaryotic integrins andinvolve divalent cation binding EF-hand and MIDAS motifs. Despite thelow level of identity between ClfA and ClfB, both proteins bindfibrinogen (on different chains) by a mechanism that is susceptible toinhibition by divalent cations, despite not sharing obvious metalbinding motifs.

Other fibrinogen binding proteins are disclosed in co-pending U.S.patent application Ser. No. 09/200,650, incorporated herein byreference. This application discloses isolated fibrinogen bindingproteins ClfB, SdrC, SdrD and SdrE as well as antibodies to the proteinsand diagnostic kits that include the proteins or the antibodies. Alsoclaimed are a method of preventing a S. aureus infection that includesadministering to the patient an effective amount of ClfB, SdrC, SdrD,SdrE, or a binding fragment thereof and a method of inducing animmunological response comprising administering to a patient apharmaceutical composition that includes ClfB, SdrC, SdrD, SdrE, or anactive fragment thereof.

ClfB has a predicted molecular weight of approximately 88 kDa and anapparent molecular weight of approximately 124 kDa. ClfB is a cell-wallassociated protein and binds both soluble and immobilized fibrinogen. Inaddition, ClfB binds both the alpha and beta chains of fibrinogen andacts as a clumping factor. The ClfB protein has been demonstrated to bea virulence factor in experimental endocarditis.

The SdrC, SdrD and SdrE proteins are related in primary sequence andstructural organization to the ClfA and ClfB proteins and are localizedon the cell surface. The SdrC, SdrD and SdrE proteins are cellwall-associated proteins, having a signal sequence at the N-terminus andan LPXTG (SEQ ID NO: 2) motif, hydrophobic domain and positively chargedresidues at the C-terminus. Each also has an SD repeat containing regionR of sufficient length to allow efficient expression of the ligandbinding domain region A on the cell surface. With the A region of theSdrC, SdrD and SdrE proteins located on the cell surface, the proteinscan interact with proteins in plasma, the extracellular matrix or withmolecules on the surface of host cells. They share some limited aminoacid sequence similarity with ClfA and ClfB. Additionally, SdrC, SdrDand SdrE also exhibit cation-dependent ligand binding to extracellularmatrix proteins. For example, SdrC binds vitronectin and SrdE binds bonesialoprotein (BSP).

It has been discovered that in the A region of SrdC, SrdD, SrdE, ClfAand ClfB there is a highly conserved amino acid sequence that can beused to derive a consensus TYTFTDYVD (SEQ ID NO: 3) motif. The motif canbe used in multicomponent vaccines to impart broad spectrum immunity tobacterial infections, and also can be used to produce monoclonal orpolyclonal antibodies that impart broad spectrum passive immunity. In analternative embodiment, any combination of the variable sequence motifderived from the Sdr and Clf protein families, (T/l) (Y/F) (TN) (F) (T)(D/N) (Y) (V) (D/N), can be used to impart immunity or produceprotective antibodies.

ClfB, SdrC, SdrD and SdrE thus share a common consensus TYTFTDYVD (SEQID NO: 3) motif which overlaps the ligand binding/Ca2+ binding region ofClfA. Therefore the proteins interact with fibrinogen and other hostcomponents. ClfB, SdrC, SdrD and SdrE subdomains, depending on theprotein, include subdomains A and B1-B5. Other information regardingextracellular matrix binding proteins has been disclosed in U.S.application Ser. No. 09/200,650, incorporated herein by reference.

V. Elastin-Binding MSCRAMMs

The primary role of elastin is to confer the property of reversibleelasticity to tissues and organs (Rosenbloom, J., et al., FASEB J.,7:1208-1218, 1993). Elastin expression is highest in the lung, skin andblood vessels, but the protein is widely expressed in mammalian hostsfor S. aureus. S. aureus binding to elastin was found to be rapid,reversible, of high affinity and ligand specific. Furthermore, a 25 kDacell surface elastin binding protein (EbpS) was isolated and proposed tomediate S. aureus binding to elastin-rich host ECM. EbpS binds to aregion in the N-terminal 30 kDa fragment of elastin.

PCT/US97/03106 discloses the gene sequences for an elastin bindingprotein. DNA sequence data disclosed indicates that the ebps openreading frame consists of 606 bp, and encodes a novel polypeptide of 202amino acids. EbpS protein has a predicted molecular mass of 23,345daltons and pI of 4.9. EbpS was expressed in E. coli as a fusion proteinwith polyhistidine residues attached to the N-terminus. A polyclonalantibody raised against recombinant EbpS interacted specifically withthe 25 kDa cell surface EbpS and inhibited staphylococcal elastinbinding. Furthermore, recombinant EbpS bound specifically to immobilizedelastin and inhibited binding of Staphylococcus aureus to elastin. Adegradation product of recombinant EbpS lacking the first 59 amino acidsof the molecule and a C-terminal fragment of CNBr-cleaved recombinantEbpS, however, did not interact with elastin. These results stronglysuggest that EbpS is the cell surface molecule mediating binding ofStaphylococcus aureus to elastin. The finding that some constructs ofrecombinant EbpS do not interact with elastin suggests that the elastinbinding site in EbpS is contained in the first 59 amino acids of themolecule.

Several independent criteria indicate that EbpS is the surface proteinmediating cellular elastin binding. First, rEbpS binds specifically toimmobilized elastin and inhibits binding of S. aureus cells to elastinin a dose dependent manner. These results establish that EbpS is anelastin binding protein that is functionally active in a soluble form.Second, an antibody raised against rEbpS recognizes a 25 kDa proteinexpressed on the cell surface of S. aureus cells. In addition to thesize similarity and antibody reactivity, further evidence that this 25kDa protein is cell surface EbpS is provided by the experiment showingthat binding of the 25 kDa protein to immobilized anti-rEbpS IgG isinhibited in the presence of excess unlabeled rEbpS. Finally, Fabfragments prepared from the anti-rEbpS antibody, but not from itspre-immune control, inhibit binding of S. aureus to elastin. This resultsuggests that the topology of surface EbpS is such that the elastinbinding site is accessible to interact with ligands (i.e. elastin andthe anti-rEbpS Fab fragment) and not embedded in the cell wall ormembrane domains. The composite data demonstrate that EbpS is the cellsurface protein responsible for binding S. aureus to elastin.

The present and previous findings suggest the existence of afunctionally active 40 kDa intracellular precursor form of EbpS thatrequires processing at the C-terminus prior to surface expression. Thisnotion is based on the following observations: i) there exists anintracellular 40 kDa elastin binding protein that is never detectedduring cell surface labeling experiments, ii) the 25 kDa EbpS and the 40kDa elastin binding protein have an identical N-terminal sequence, andiii) a single gene exists for EbpS. Because the size of the ebps openreading frame is not sufficient to encode a 40 kDa protein, at first theinventors disregarded this hypothesis. However, their studies with rEbpSdemonstrated that although the actual size of the recombinant protein is26 kDa, it migrates aberrantly as a 45 kDa protein in SDS-30 PAGE. Thisfinding suggests that full length native EbpS, with a predicted size of23 kDa, may be migrating in SDS-PAGE as the 40 kDa intracellularprecursor, and that the 25 kDa surface form of EbpS is actually asmaller form of the molecule processed at the C-terminus. Although EbpSlacks an N-terminal signal peptide and other known sorting and anchoringsignals, this proposed intracellular processing event may explain somequestions regarding how EbpS is targeted to the cell surface. In fact,C-terminal signal peptides have been identified in several bacterialproteins (Fath, M. J. and Kolter, R., Microbiol. Rev., 57:995-1017,1993) and alternative means of anchoring proteins to the cells surfacehave been reported in gram positive bacteria (Yother, J. and White, J.M., J. Bacteriol., 176:2976-2985, 1994).

Using overlapping EbpS fragments and recombinant constructs, the elastinbinding site in EbpS was mapped to the amino terminal domain of themolecule (PCT/US97/03106). Overlapping synthetic peptides spanning aminoacids 14-34 were then used to better define the binding domain. Amongthese, peptides corresponding to residues 14-23 and 18-34 specificallyinhibited elastin binding by more than 95%. Common to all activesynthetic peptides and proteolytic and recombinant fragments of EbpS isthe hexameric sequence ¹⁸Thr-Asn-Ser-His-Gln-Asp²³. Further evidencethat this sequence is important for elastin binding was the loss ofactivity when Asp²³ was substituted with Asn in the synthetic peptidecorresponding to residues 18-34. However, the synthetic hexamer TNSHQDby itself did not inhibit staphylococcal binding to elastin. Thesefindings indicate that although the presence of the TNSHQD sequence isessential for EbpS activity, flanking amino acids in the N- orC-terminal direction and the carboxyl side chain of Asp²³ are requiredfor elastin recognition.

VI. MHC II—Analogous Proteins, (MAP)

In addition to fibrinogen, fibronectin, collagen and elastin, S. aureusstrains associate with other adhesive eukaryotic proteins, many of whichbelong to the family of adhesive matrix proteins, such as vitronectin.(Chatwal, G. S., et al., Infect. Immun., 55:1878-1883, 1987). U.S. Pat.No. 5,648,240, incorporated herein by reference, discloses a DNA segmentcomprising a gene encoding a S. aureus broad spectrum adhesin that has amolecular weight of about 70 kDa. The adhesin is capable of bindingfibronectin or vitronectin and includes a MHC II mimicking unit of about30 amino acids. Further analyses of the binding specificities of thisprotein reveal that it functionally resembles an MHC II antigen in thatit binds synthetic peptides. Thus, in addition to mediating bacterialadhesion to ECM proteins, it may play a role in staphylococcalinfections by suppressing the immune system of the host. The patentfurther claims a recombinant vector that includes the specified DNAsequence, a recombinant host cell transformed with the vector, and DNAwhich hybridizes with the DNA of specified sequence. Also disclosed is acomposition that includes a protein or polypeptide encoded by thespecified DNA sequence and a method of inducing an immune response in ananimal that includes administering an immunogenic composition thatincludes the encoded protein or polypeptide. A method of making a MHC IIantigen protein analog comprising the steps of inserting the specifiedDNA sequence in a suitable expression vector and culturing a host celltransformed with the vector under conditions to produce the MHC IIantigen protein analog is additionally claimed in the patent.

VII. SDR Proteins from Staphylococcus epidermidis

Staphylococcus epidermidis, a coagulase-negative bacterium, is a commoninhabitant of human skin and a frequent cause of foreign-bodyinfections. Pathogenesis is facilitated by the ability of the organismto first adhere to, and subsequently to form biofilms on, indwellingmedical devices such as artificial valves, orthopedic devices, andintravenous and peritoneal dialysis catheters. Device-related infectionsmay jeopardize the success of medical treatment and significantlyincrease patient mortality. Accordingly, the ability to develop vaccinesthat can control or prevent outbreaks of S. epidermidis infection is ofgreat importance, as is the development of means that can prevent ortreat infection from a broad spectrum of bacteria, including bothcoagulase-positive and coagulase negative bacteria.

Three Sdr (serine-aspartate (SD) repeat region) proteins that areexpressed by S. epidermidis have been designated as SdrF, SdrG and SdrH,and the amino acid sequences of these proteins and their nucleic acidsequences are disclosed in co-pending U.S. patent application of Fosteret al. which is based on U.S. provisional application Ser. Nos.60/098,443 and 60/117,119. All of these applications are incorporatedherein by reference.

In accordance with the present invention, the donor selection and donorstimulation methods described herein can also be performed with regardto the SdrF, SdrG or an SdrH protein. In these methods, individuals maybe identified and selected who have higher than normal antibody titersto the SdrF, SdrG or an SdrH proteins, and a donor plasma pool can beprepared which will have higher than normal titers to one or more ofthese proteins. Accordingly, donor plasma can be prepared in accordancewith the present invention which will be useful in methods to prevent ortreat infection from coagulase-negative staphylococcal infections suchas those associated with S. epidermidis.

VIII. Proteins and Peptides with Substantial Homology or EquivalentFunction to Those Described Herein

Donor plasma pools can be screened or stimulated as desired, with fullsequence proteins, peptides, protein or peptide fragments, isolatedepitopes, fusion proteins, or any alternative which binds to the targetECM, whether in the form of a wild type, a site-directed mutant, or asequence which is substantially homologous thereto.

When used in conjunction with amino acid sequences, the term“substantially similar” means an amino acid sequence which is notidentical to published sequences, but which produces a protein orpeptide having the same functionality and activities, either because oneamino acid is replaced with another similar amino acid, or because thechange (whether it be substitution, deletion or insertion) does notsubstantially effect the active site of the protein. Two amino acidsequences are “substantially homologous” when at least about 70%,(preferably at least about 80%, and most preferably at least about 90 or95%) of the amino acids match over the defined length of the sequences.

It should also be understood that each of the MSCRAMM polypeptides ofthis invention may be part of a larger protein. For example, a ClfApolypeptide of this invention may be fused at its N-terminus orC-terminus to a ClfB polypeptide, or to a non-fibrinogen bindingpolypeptide or combinations thereof. Polypeptides which may be usefulfor this purpose include polypeptides derived any of the MSCRAMMproteins, and serotypic variants of any of the above.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according toTable 1. In keeping with standard polypeptide nomenclature (J. Biol.Chem., 243:3552-3559, 1969), abbreviations for amino acid residues areshown in Table I. It should be understood by one skilled in the art thatthe codons specified in Table 1 are for RNA sequences. The correspondingcodons for DNA have a T substituted for U.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties which can stimulate the production of a substantiallysimilar antibody. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, corresponding DNA sequences which encode said peptides orantibodies against said peptides without appreciable loss of thebiological utility or activity of the donor plasma pool immunoglobulinthat is recovered. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCGGCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU GAC GAU Glutamicacid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GCG GGGGGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG GUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It is understood in the art that the substitution of like amino acidscan be made effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+1.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

The following non-classical amino acids may be incorporated in thepeptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3—carboxylate (Kazmierski et al., J. Am.Chem. Soc., 113:2275-2283, 1991); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett.,1991); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, Ph.D.Thesis, University of Arizona, 1989);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al, J.Takeda Res. Labs., 43:53-76, 1989); β-carboline (D and L) (Kazmierski,Ph.D. Thesis, University of Arizona, 1988); HIC (histidine isoquinolinecarboxylic acid) (Zechel et al, Int. J. Pep. Protein Res., 43, 1991);and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al, J. Org. Chem., 50:5834-5838, 1985);β-sheet inducing analogs (Kemp et al., Tetrahedron Lett., 29:5081-5082,1988); β-turn inducing analogs (Kemp et al., Tetrahedron Lett.,29:5057-5060, 1988); alpha-helix inducing analogs (Kemp et al.,Tetrahedron Lett., 29:4935-4938, 1988); ?-turn inducing analogs (Kemp etal., J. Org. Chem., 54:109:115, 1989); and analogs provided by thefollowing references: Nagai and Sato, Tetrahedron Lett., 26:647-650(1985); DiMaio et al., J. Chem. Soc. Perkin Trans., p. 1687 (1989); alsoa Gly-Ala turn analog (Kahn et al., Tetrahedron Lett., 30:2317, 1989);amide bond isostere (Jones et al., Tetrahedron Lett., 29:3853-3856,1988); tetrazol (Zabrocki et al., J. Am. Chem. Soc., 110:5875-5880,1988); DTC (Samanen et al., Int. J. Protein Pep. Res., 35:501:509,1990); and analogs taught in Olson et al., J. Am. Chem. Sci.,112:323-333 (1990) and Garvey et al., J. Org. Chem., 56:436 (1990).Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

IX. Preparation of Purified Immunoglobulin

In one embodiment, purified immunoglobulin (A, D, E, or G) is preparedthat has a high titer of antibodies to the selected adhesins. The term“high titer” in this context means the presence of an antibody in anamount which is 2-fold or greater, e.g., up to 10-20 more times higherthan that found in a normal population of 100 random samples of blood.

The blood product can be prepared by (i) selection and purification ofthe immunoglobulin of a donor which has naturally high titers ofantibodies to the selected adhesins, (ii) the combination of donorimmunoglobulin from several individuals which have a high titer ofantibodies to one or more of the selected adhesins, to produce thedesired composite profile; or (iii) stimulation of the desiredantibodies in one or more donors to form the desired composite antibodyprofile by exposing the donor to the selected antigens and obtainingblood sample of the exposed donor after sufficient time to produce andaccumulate the resulting immunoreactive antibodies. The first twoembodiments are referred to as “donor select” programs and the third isreferred to as a “donor stimulation” program.

Donor Stimulation

Using the peptide antigens described herein, the present invention alsoprovides methods of stimulating high antibody levels in a donor, whichincludes administering to an animal, for example a human, apharmaceutically-acceptable composition comprising an immunologicallyeffective amount of an MSCRAMM-derived peptide composition. Thecomposition can include partially or significantly purifiedMSCRAMM-derived peptide epitopes, obtained from natural or recombinantsources, which proteins or peptides may be obtainable naturally oreither chemically synthesized, or alternatively produced in vitro fromrecombinant host cells expressing DNA segments encoding such epitopes.Smaller peptides that include reactive epitopes, such as those betweenabout 30 and about 100 amino acids in length will often be preferred.The antigenic proteins or peptides may also be combined with otheragents, such as other staphylococcal or streptococcal peptide or nucleicacid compositions, if desired. The composition may also includestaphylococcal produced bacterial components such as those discussedabove, obtained from natural or recombinant sources, which proteins maybe obtainable naturally or either chemically synthesized, oralternatively produced in vitro from recombinant host cells expressingDNA segments encoding such peptides.

Further means contemplated by the inventors for generating an immuneresponse in an animal includes administering to the animal, or humansubject, a pharmaceutically-acceptable composition comprising animmunologically effective amount of a nucleic acid composition encodinga peptide epitope, or an immunologically effective amount of anattenuated live organism that includes and expresses such a nucleic acidcomposition. Antigenic functional equivalents of the proteins andpeptides described herein also fall within the scope of the presentinvention. Antigenically functional equivalents, or epitopic sequences,may be first designed or predicted and then tested, or may simply bedirectly tested for cross-reactivity.

In the case of preventing bacterial adhesion, the preparation ofepitopes which produce antibodies which inhibit the interaction of aspecific gene product or proteoglycans which are structurally similar tothe specific gene product are particularly desirable.

The identification or design of suitable MSCRAMM epitopes, and/or theirfunctional equivalents, suitable for use in immunoformulations,vaccines, or simply as antigens (e.g., for use in detection protocols),is a relatively straightforward matter. For example, one may employ themethods of Hopp, as enabled in U.S. Pat. No. 4,554,101, incorporatedherein by reference, that teaches the identification and preparation ofepitopes from amino acid sequences on the basis of hydrophilicity. Theamino acid sequence of these “epitopic core sequences” may then bereadily incorporated into peptides, either through the application ofpeptide synthesis or recombinant technology.

Plasmapheresis

The term plasmapheresis describes a technique in which blood is removedfrom an animal, separated into its cellular and plasma components, thecells are then returned to the animal, and the plasma retained. Largevolume plasmapheresis requires the removed plasma to be replaced by asuitable fluid, and when this is done, the technique is often known asplasma exchange. Any components found in plasma can be removed by plasmaexchange. Plasma exchange is the method still in use at most blood banksand public donation centers in the United States. Plasma extracted thisway for commercial sale is available for use in a preferred embodimentof this invention.

During plasma donation, it is necessary to replace the fluid taken toprevent circulatory collapse. In most circumstances, the osmotic effectof the plasma needs to be replaced. A 5% solution of human albuminobtained from donor blood is a safe and effective replacement. It isstandard practice in the medical community to add 2 ml of KCl solutionand 2 ml of 10% calcium gluconate solution to the albumin. Most plasmaexchange units replace every 2 liters of plasma removed with 1.5 litersof human albumin solution and 0.5 liters of normal saline.

The methods currently in use for plasma separation are centrifugationand filtration. The technique of U.S. Pat. No. 5,548,066 may be used toprepare the donor plasma pool if it is not commercially available, andis incorporated by reference herein. First, a plurality of blood donorsare identified. These donors are mature mammals, typically mammals ofthe same species for which the serum will be employed. Where a specificailment is to be treated or prevented, such as mastitis in mammals orother diseases caused by staphylococcal bacteria such as S. aureus, itis preferred that the donors have been exposed either naturally orthrough immunization to the causative organism or some antigenic portionthereof. Further, to achieve a consistent serum product, it is preferredthat the donor group be relatively large. It is preferred to use humanhosts to prepare the donor plasma pools. Once the donors have beenidentified, blood is drawn from the donors. Since the serum is refineddirectly from the blood, it is desired to obtain the maximum quantity ofblood to thus obtain the maximum quantity of serum. For humans, anestablished limit of blood is drawn periodically over time.

It is preferred to identify and maintain a consistent donor group byrepeated drawing of smaller quantities of blood, for example, drawing ofblood once a month from humans. The frequency of the drawing will ofcourse influence the quantity which may be safely drawn. In general, itis desired to draw the maximum amount of blood over the course of timewithout causing detriment to the health of the donor. This may dictatedrawing small amounts with great frequency, or the maximum amountpossible at a reduced frequency, depending upon the particular species.The blood volume of the donor may be estimated by standard formulasavailable from the Center for Disease Control.

The health of the donor is of course a consideration in this process iflong-term bleeding is desired. Before donating, the donor will bechecked for general good health, and if the donor is in poor health thebleeding may be deferred until the next scheduled date. Beyond this, itis preferred that long-term health records be kept, preferably includingmore detailed information. In this regard, it is noted that productionquantities of the present serum is a good indicator of the health of thedonor.

Typically, the serum is separated from the blood of the donor andconsists of material from the immune system. In one method, detailedrecords are kept for the amount of serum produced from the blood as ayield percentage, such as 7 liters of serum from 14 liters of bloodprovides a yield of 50%. In the preferred method, records of the yieldpercentage are kept for each donor for each bleeding. These percentagesmay then be used to determine if the donor should be bled at the nextscheduled time. In particular, if the action to be taken is expressed asa function of yield percentages, a guideline may be expressed asfollows: yield percentage </=30%, rest; 31-35%, caution; 36-59%, normal;60-64%, caution; >/=65%, rest. As may be seen, the donor is not bled ifthe serum yield is above or below the normal range. Such a yieldpercentage may indicate an underlying ailment. The subject may be bled,possibly in a reduced amount, in the caution ranges, depending upon thedonor's history and/or further examination. In this regard, it has beenfound that a small percentage of individuals consistently produce yieldpercentages around 60-62%.

The method of blood and plasma collection is generally standard and wellto known to those of skill in the art. Any method can be used thatachieves the desired results. Once the blood has been collected, it issubjected to procedures for extracting the desired components. A firstimportant step in this process is to permit each vessel of collectedblood to sit at room temperature at least until substantial clotting hasoccurred, usually one hour. During this period the blood moves from bodytemperature to room temperature, and is exposed to air. This exposure toair permits the fibrinogen to change into fibrin, causing clotting ofthe blood.

This clotting period is an important aspect of serum retrieval. Theclotting provides a rough separation of the cellular material from theliquid. Additionally, while the exact mechanism is not known, it isbelieved that the clotting period causes white blood cells to die and,for a percentage of such cells, to burst or rupture such that thechemical material, including antibody, therein is released from thecells. It is believed that this material remains within the serum andacts to provide “information” to the immune system of the recipient ofthe serum. This “information” may help to “program” white blood cellsfor particular microorganisms, similar to providing them with a memoryof the microorganism, such that the white blood cells of the recipientrespond quickly, and in a manner similar to a subject which has beenvaccinated or is immune.

This period of non-refrigeration also causes a rough filtering of thecollected blood. In particular, the clotted blood with the relativelyheavy red blood cells will fall toward the bottom of the vessel, whilethe liquid plasma, immunoglobulins and chemical material will be pushedtoward the top. To assist in this process, and a process describedbelow, it is preferred that the collection vessel be tall and thin,having proportions similar to a standard test tube.

The liquid portion obtained at this stage is raw serum which, afterbeing filtered and sterilized, can impart immunity. Further steps areoptionally carried out. However, to increase the yield, various othersteps prior to filtration are preferred.

A first of these steps, after the collected blood has had sufficienttime to clot, is refrigeration to approximately 20-60° C. Thisrefrigeration reduces the temperature of the blood from room temperatureto the refrigeration temperature. Such cooling of course prevents growthof bacteria, mold, etc. Additionally, during this cooling the clottedblood settles further, and the clotted blood

contracts. This contraction (and possibly the cooling) may cause afurther percentage of the white blood cells to rupture. Additionally,the contraction of the clotted blood serves to express from the clotimmunoglobulins and chemical materials which have been trapped therein.This refrigeration should last at least until the blood has achieved therefrigeration temperature, and preferably for about 14-18 hours, orovernight.

A second preferred step is physical pressing of the clotted blood. Thispressing is believed to cause yet more rupturing of white cells, thusyielding even more of the transfer factor. Additionally, in a mannersimilar to the cooling contraction, the pressing serves to forceimmunoglobulins and transfer factor from the clot.

The preferred method of pressing is to insert a sterile weight into therefrigerated vessel of collected blood. For example, a cylinder having aclose sliding fit within the vessel and a weight of approximately twopounds. As may be envisioned, the liquid material will flow about thecylinder until the cylinder has come to rest upon the clotted bloodsettled at the bottom of the vessel. It is preferred that the pressingweight be maintained in place for about 6-24 hours.

It is noted that the pressing can serve as a first active filtrationstep. The close fit of the weight serves to separate the liquid rawserum above and the solid material below, although a precision fit ofthe weight in the vessel is not required. Since this may serve as afirst, rough, filtration step, it may conveniently be used to determinethe quantity of raw serum produced for calculation of the yieldpercentage. Specifically, noting the height of the column of raw serumand knowing the diameter of the vessel provides the volume of raw serumproduced.

At this point the filtering process proper begins. This furtherprocessing includes filtration to remove all cellular material. Thisfiltration is achieved in multiple steps. The first filtration step is agross filtering. This may be achieved simply by pouring the contents ofthe vessel into a collection vat while holding a screen over the openingin the collection vessel. Where the high-yield steps of refrigerationand pressing have been used, the pressing cylinder still within thevessel may act in conjunction with the screen to filter, and the screenmay mainly filter out the cylinder itself. Where these high-yield stepshave not been taken, a finer filter screen may be desired. The clottedcells remaining within the vessel are properly disposed of, and thevessel sterilized for later use.

This is a preferred point for combining the serum from different donors.It is noted, however, that samples from multiple donors can be combinedat any point subsequent to the initial gross filtration step.

The raw serum may still contain a large amount of cells and cellulardebris. As the next filtration step, the reclaimed liquid is then placedinto a continuous flow centrifuge. For example, the liquid may be placedin a Sharples AS16NF continuous flow centrifuge, which will operate atapproximately 13,000 to 15,000 rpm. The liquid is drawn off during thisprocess while yet more of the cells and cellular debris is removed.

Following the isolation of the plasma, the antibodies are purified awayfrom other cell products. This can be accomplished by a variety ofprotein isolation procedures, known to those skilled in the art ofimmunoglobulin purification, such as ion exchange, affinitypurification, etc. Means for preparing and characterizing antibodies arewell known in the art. For example, serum samples can be passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the Abs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The Ab is then eluted with3.5 M magnesium chloride. Abs purified in this way can then tested forbinding activity by, for example, an isotype-specific ELISA andimmunofluorescence staining assay of the target cells.

In an alternative embodiment, the liquid is instead subjected to afurther filtration step. This further step actually consists of severalsub-steps, with the liquid being passed through several filters ofprogressively finer gauge. In particular, the liquid is passed throughat least a 0.65 micron filter, then a 0.2 micron nominal filter, andthen through a 0.2 micron absolute filter. By passing the liquid throughthe 0.2 nominal filter first, most of the bacteria, mold, and fibrinwill be removed prior to passing through the 0.2 absolute filter.

At this point the liquid has had essentially all solid cellular materialremoved. The chemical materials and immunoglobulins, however, remain inthe liquid, which is referred to as clarified serum.

The clarified serum can be used (after sterilization described below) asthe final serum. However, it is preferred that the clarified serum beconcentrated. This concentration reduces the volume and thus reduces theamount of material which must be shipped. Additionally, certainrecipients, such as infant mammals, can not accept a large quantity ofmedication intravenously due to a lack of capacity. As such,concentration permits a full dosage of the serum to be administered. Theconcentration is preferably performed by repeated ultra-filtration toremove water molecules, as is known in the art. Such filtration has acut-off filter of between 10,000 and 100,000 mol. wt. During thisprocess, samples of the clarified serum may be taken to determine if theserum has been sufficiently concentrated. It is preferred that the finalserum be concentrated to about 2 to 6 times the clarified serum, andmost preferably 2 to 4 times.

Determination of the concentration level is made by testing the amountof IgG (or other immunoglobulin) within the serum. An initial test maybe made of the clarified serum, and this result compared with the testsmade upon the serum during the ultra-filtration process. For example, ifthe initial test results in the clarified serum having an IgGconcentration of 1 g/100 ml, then the concentration process may bestopped when later tests report an IgG concentration of between about2-6 g/100 ml, and preferably about 3 g/100 ml. The determination of theIgG amount may be made by the radial immunodiffusion test. However, itis preferred that serum protein electrophoresis be performed on thewhole serum to obtain an entire gamma globulin result. This is believedto be more accurate, and provides a clear indication of the IgG level.Once the concentration process has been completed the concentratedunsterilized serum is bottled or packaged using standard procedures.

Upon completion of the concentration and packaging process, the resultis unsterilized serum. The next step is to sterilize the serum. Whilethis sterilization is effected, it is important that the unsterilizedserum not be denatured. To provide sterilization without denaturing, theunsterilized serum is frozen to a hard freeze condition. For theunsterilized serum, this is approximately −29° C. (−21° F.). While stillfrozen, the material is then subjected to sufficient gamma irradiationthat the material is sterilized, but is not denatured. This level mayvary among various species, but may be determined without undueexperimentation. It is important that the material be sufficiently cold(hard frozen) such that the material remains frozen during theirradiation step, otherwise denaturing will occur. It is for this reasonthat the material is frozen to the relatively low temperature. If it isfound that if the irradiation process is sufficiently short, orrefrigeration is provided during irradiation, then a higher temperature(though still below freezing) could be tolerated.

At this point the final serum has been obtained, although it is frozen.The packages of the serum are thus placed in refrigeration and allowedto thaw to the refrigeration temperature, where they are stored untiluse.

After administration, the serum has been found to provide cellularimmunity similar to a vaccine, and can be used with or without theintroduction of the virulent. In general, the present serum shouldprovide protection against bacteria for which the donor group hasimmunity. In humans, a wide variety of vaccination uses are possible,including general vaccination for individuals with impaired immunity,such as is caused by diabetes, and vaccination for individuals preparingto undergo surgery due to the of nosocomial infection. In addition tohumans, the inventive serum should also be of utility for many mammals,such as farm and domestic mammals and humans. For cattle, one particularuse would be to avoid bovine mastitis, a common ailment which costs thedairy industry millions of dollars per year.

X. Uses for MSCRAMM and Antibody Compositions

The immunotherapeutic product of the present invention is a purified andconcentrated extract of plasma, or serum from a purified donor pool. Theserum contains antibodies released from the white blood cells in theextracted blood, and possibly other chemical materials present in theextracted blood. This serum is believed to provide information which is“read” by the immune system of the recipient to provide an extendedperiod of immunity, typically on the order of six to eight weeks.Purified donor plasma pools can be used for the treatment of wounds, forblocking protein receptors or for immunization (vaccination).

The plasma pools comprise antibodies which are useful for interferingwith the initial physical interaction between a pathogen and mammalianhost responsible for infection, such as the adhesion of bacteria,particularly gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block protein-mediated mammalian cell invasion; to blockbacterial adhesion between mammalian extracellular matrix proteins andbacterial proteins that mediate tissue damage; and, to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or surgical techniques.

In general, both poly- and monoclonal antibodies against MSCRAMMpeptides may be used in a variety of embodiments. For example, they maybe employed in antibody cloning protocols to obtain cDNAs or genesencoding the peptides discussed herein or related proteins. They mayalso be used in inhibition studies to analyze the effects ofMSCRAMM-derived peptides in cells or animals. Anti-MSCRAMM epitopeantibodies will also be useful in immunolocalization studies to analyzethe distribution of MSCRAMMs during various cellular events, forexample, to determine the cellular or tissue-specific distribution ofthe MSCRAMM peptides under different physiological conditions. Aparticularly useful application of such antibodies is in purifyingnative or recombinant MSCRAMMS, for example, using an antibody affinitycolumn. The operation of all such immunological techniques will be knownto those of skill in the art in light of the present disclosure.

Immunological compositions, including vaccine, and other pharmaceuticalcompositions containing the selected donor pool plasma concentrate areincluded within the scope of the present invention. The combination ofimmunoglobulins against binding proteins, or active or antigenicfragments thereof, or fusion proteins thereof, can be formulated andpackaged, alone or in combination with other antibodies, using methodsand materials known to those skilled in the art for vaccines. Theimmunological response may be used therapeutically or prophylacticallyand may provide passive immunity.

XI. Preparation of Proteins and Antibodies

The skilled reader can employ conventional molecular biology,microbiology, and recombinant DNA techniques to prepare the proteins,peptides, and antibody compositions described herein. Such techniquesare explained fully in the literature. See, e.g., Sambrook et al,“Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols inMolecular Biology” Volumes I-III (Ausubel, R. @-I ed., 1994); “CellBiology: A Laboratory Handbook” Volumes I-III (J. E. Celis, ed., 1994);“Current Protocols in Immunology” Volumes I-III ([Coligan, J. E., ed.,1994); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds., 1985); “TranscriptionAnd Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal CellCulture” (R. I. Freshney, ed, (1986); “Immobilized Cells And Enzymes(IRL Press, 1986); B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

The antibody obtained through this invention may be labeled directlywith a detectable label for identification and quantification ofstaphylococcal bacterial such as S. aureus, S. epidermidis, etc. Labelsfor use in immunoassays are generally known to those skilled in the artand include enzymes, radioisotopes, and fluorescent, luminescent andchromogenic substances including colored particles such as colloidalgold and latex beads. Suitable immunoassays include enzyme-linkedimmunosorbent assays (ELISA).

Alternatively, the antibody can be labeled indirectly by reaction withlabeled substances that have an affinity for immunoglobulin, such asprotein A or G or second antibodies. The antibody may be conjugated witha second substance and detected with a labeled third substance having anaffinity for the second substance conjugated to the antibody. Forexample, the antibody may be conjugated to biotin and theantibody-biotin conjugate detected using labeled avidin or streptavidin.Similarly, the antibody may be conjugated to a hapten and theantibody-hapten conjugate detected using labeled anti-hapten antibody.These and other methods of labeling antibodies and assay conjugates arewell known to those skilled in the art. Antibodies to the bindingproteins may also be used in production facilities or laboratories toisolate additional quantities of the protein, such as by affinitychromatography.

In another identification embodiment, microliter plates pre-treated withpoly-L-lysine are used to bind one of the target cells to each well, thecells are then fixed, e.g. using 1% glutaraldehyde, and the antibodiesare tested for their ability to bind to the intact cell. In addition,FACS, immunofluorescence staining, idiotype specific antibodies, antigenbinding competition assays, and other methods common in the art ofantibody characterization may be used in conjunction with the presentinvention to identify preferred donors.

Humanized antibodies are antibodies of animal origin that have beenmodified using genetic engineering techniques to replace constant regionand/or variable region framework sequences with human sequences, whileretaining the original antigen specificity.

Such antibodies are commonly derived from rodent antibodies withspecificity against human antigens. Such antibodies are generally usefulfor in vivo therapeutic applications. This strategy reduces the hostresponse to the foreign antibody and allows selection of the humaneffector functions.

The techniques for producing humanized immunoglobulins are well known tothose of skill in the art. For example, U.S. Pat. No. 5,693,762discloses methods for producing, and compositions of, humanizedimmunoglobulins having one or more complementarily determining regions(CDR's). When combined into an intact antibody, the humanizedimmunoglobulins are substantially non-immunogenic in humans and retainsubstantially the same affinity as the donor immunoglobulin to theantigen, such as a protein or other compound containing an epitope.Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present invention includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobin preparations and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

U.S. Pat. No. 5,565,332 describes methods for the production ofantibodies, or antibody fragments, which have the same bindingspecificity as a parent antibody but which have increased humancharacteristics. Humanized antibodies may be obtained by chainshuffling, perhaps using phage display technology, in as much as suchmethods will be useful in the present invention the entire text of U.S.Pat. No. 5,565,332 is incorporated herein by reference.

XII. Production of High Titer MSCRAMM-Specific IgG from BiologicalFluids Via Affinity Purification

In accordance with the present invention, it is also possible to utilizemodes of affinity isolation and purification in order to produce hightiter MSCRAMM-specific immunoglobulins from biological fluids such asblood or plasma. In the preferred modes of this aspect of the invention,recombinant or wild-type/native MSCRAMMs can be covalently coupled to asubstrate or resin, such as Sepharose™ or agarose, to form an affinitymatrix. The MSCRAMM affinity matrix can be used to selectively isolateantibodies from serum, plasma, or other biological fluids. In thepreferred embodiment, the biological fluid is passed over the MSCRAMMaffinity matrix, and the matrix is then washed to removenon-specifically bound antibodies. The washed matrix is then subjectedto conditions, such as low pH or high salt, so that MSCRAMM specificantibodies remaining on the matrix are eluted. The anti-MSCRAMM titer ofthe eluted material will be considerably higher than that of theoriginal biological fluid, and the eluted material can then be utilizedin the same manner as the other donor-selected or donor-stimulatedcompositions of the present invention.

XIII. Pharmaceutical Compositions

Pharmaceutical compositions for immunization of donors containing theMSCRAMM proteins, nucleic acid molecules, antibodies, or fragmentsthereof may be formulated in combination with a pharmaceutical carriersuch as saline, dextrose, water, glycerol, ethanol, other therapeuticcompounds, and combinations thereof. The formulation should beappropriate for the mode of administration. Suitable methods ofadministration include, but are not limited to, oral, anal, vaginal,intravenous, intraperitoneal, intramuscular, subcutaneous, intranasaland intradermal administration.

The preferred route is by intravenous administration.

The pharmaceutical composition for treatment of any of the conditionsdescribed herein, should comprise, in a pharmaceutically acceptableexcipient, an effective amount of immunoglobulin to treat or prevent thetarget disorder.

Compositions which contain immunoglobulins as active ingredients arewell understood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions, however, solidforms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.The active therapeutic ingredient is often mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient. The therapeutic donor immunoglobulin pool compositions areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for humans, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of MSCRAMM binding capacity desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or moreintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations of ten nanomolar to ten micromolar in the blood arecontemplated.

The immunological compositions, such as vaccines, and otherpharmaceutical compositions can be used alone or in combination withother blocking agents to protect against human and animal infectionscaused by staphylococcal bacterial including S. aureus and others. Inparticular, the compositions can be used to protect humans againstendocarditis or to protect humans or ruminants against mastitis causedby staphylococcal infections. The vaccine can also be used to protectcanine and equine animals against similar staphylococcal infections.

To enhance immunogenicity, the donor plasma pool concentrate proteinsmay be conjugated to a carrier molecule. Suitable immunogenic carriersinclude proteins, polypeptides or peptides such as albumin, hemocyanin,thyroglobulin and derivatives thereof, particularly bovine serum albumin(BSA) and keyhole limpet hemocyanin (KLH), polysaccharides,carbohydrates, polymers, and solid phases. Other protein derived ornon-protein derived substances are known to those skilled in the art. Animmunogenic carrier typically has a molecular weight of at least 1,000daltons, preferably greater than 10,000 daltons. Carrier molecules oftencontain a reactive group to facilitate covalent conjugation to thehapten. The carboxylic acid group or amine group of amino acids or thesugar groups of glycoproteins are often used in this manner. Carrierslacking such groups can often be reacted with an appropriate chemical toproduce them. Preferably, an immune response is produced when theimmunogen is injected into animals such as mice, rabbits, rats, goats,sheep, guinea pigs, chickens, and other animals, most preferably miceand rabbits. Alternatively, a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide may be sufficiently antigenic toimprove immunogenicity without the use of a carrier.

In a preferred embodiment, a donor stimulating vaccine is packaged forimmunization by parenteral (i.e., intramuscular, intradermal orsubcutaneous) administration or nasopharyngeal (i.e., intranasal)administration. The vaccine is most preferably injected intramuscularlyinto the deltoid muscle. The vaccine is preferably combined with apharmaceutically acceptable carrier to facilitate administration. Thepreferred carrier is usually water or a buffered saline, with or withouta preservative. The vaccine may be lyophilized for resuspension at thetime of administration or in solution.

The carrier to which the protein may be conjugated may also be apolymeric delayed release system. Synthetic polymers are particularlyuseful in the formulation of a vaccine to effect the controlled releaseof antigens. For example, the polymerization of methyl methacrylate intospheres having diameters less than one micron has been reported byKreuter, J., “Microcapsules and Nanoparticles in Medicine andPharmacology,” M. Donbrow, Ed., CRC Press, p. 125-148.

The amount of immunogen composition used in the production of thepolyclonal antibodies varies upon the nature of the immunogen, as wellas the animal used for immunization. The preferred dose for humanadministration is from 0.01 mg/kg to 10 mg/kg, preferably approximately1 mg/kg. Based on this range, equivalent dosages for heavier bodyweights can be determined. The dose should be adjusted to suit theindividual to whom the composition is administered and will vary withage, weight and metabolism of the individual. The vaccine mayadditionally contain stabilizers such as thimerosal(ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma ChemicalCompany, St. Louis, Mo.) or physiologically acceptable preservatives.

The production of polyclonal antibodies may be monitored by samplingblood of the immunized animal at various points following immunization.A second, booster injection, also may be given. The process of boostingand titering is repeated until a suitable titer is achieved. When adesired level of immunogenicity is obtained, the process may continue.

The compositions preferably further comprise an adjuvant. Many adjuvantsare known for use in vaccinations in animals and are readily adapted tothis composition. At this time, the only adjuvant widely used in humanshas been alum (aluminum phosphate or aluminum hydroxide). Saponin andits purified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines.

The isolated peptide can be linked to a selected amino acid sequence tomake a fusion protein. As a nonlimiting example, a fusion protein can bemade that comprises at least a first peptide of a fibronectin bindingdomain of fibronectin binding protein operatively linked to a selectedamino acid sequence, wherein the first peptide does not specificallybind to fibronectin. In preferred aspects, the first peptide is linkedto a selected carrier molecule or amino acid sequence, including, butnot limited to, keyhole limpet hemocyanin (KLH) and bovine serum albumin(BSA).

One of the important features provided by the donor stimulationembodiment of the present invention is a polyclonal sera that isrelatively homogenous with respect to the specificity of the antibodiestherein. Typically, polygonal antisera is derived from a variety ofdifferent “clones,” i.e., B-cells of different lineage. Monoclonalantibodies, by contrast, are defined as coming from antibody-producingcells with a common B-cell ancestor, hence their “mono” clonality.

When peptides are used as antigens to stimulate the production ofpolyclonal sera, one expects considerably less variation in the clonalnature of the sera than if a whole antigen were employed. Unfortunately,if incomplete fragments of an epitope are presented, the peptide mayvery well assume multiple (and probably non-native) conformations. As aresult, even short peptides can produce polyclonal antisera withrelatively plural specificities and, unfortunately, an antisera thatdoes not react or reacts poorly with the native molecule.

Polyclonal antisera according to the present invention is producedagainst peptides that are predicted to comprise whole, intact epitopes.It is believed that these epitopes are, therefore, more stable in animmunologic sense and thus express a more consistent immunologic targetfor the immune system. Under this model, the number of potential B-cellclones that will respond to this peptide is considerably smaller and,hence, the homogeneity of the resulting sera will be higher. In variousembodiments, the present invention provides for polyclonal antiserawhere the clonality, i.e., the percentage of clone reacting with thesame molecular determinant, is at least 80%. Even higher clonality—90%,95% or greater—is contemplated.

XIV. Kits

This invention also includes a kit for the identification of blood orplasma with high titers of desired antibodies. The preferred kitcontains sufficient antigen to bind substantially all of the antibody inthe sample in about ten minutes or less, or sufficient antibody whichcan target an antibody in the sample that is to be detected. The antigenor antibody in the kit, e.g., any of the MSCRAMMs or their bindingdomains as described above, is preferably immobilized on a solidsupport, and can be labeled with a detectable agent such as thosedescribed above or commonly known in the art. The kit optionallycontains a means for detecting the detectable agent. If the antigen orantibody in the kit is labeled with a fluorochrome or radioactive label,no means for detecting the agent will typically be provided, as the userwill be expected to have the appropriate spectrophotometer,scintillation counter, or microscope. If the detectable agent is anenzyme, a means for detecting the detectable agent can be supplied withthe kit, and would typically include a substrate for the enzyme insufficient quantity to detect all of the antigen-antibody complex. Onepreferred means for detecting a detectable agent is a substrate that isconverted by an enzyme into a colored product. A common example is theuse of the enzyme horseradish peroxidase with2,2′-azino-di-[3-ethyl-benzothiazoline sulfonate] (ABTS).

The invention includes a method for detecting biological samples with anelevated titer of antibodies to selected staphylococcal MSCRAMMs. Asused herein the term biological sample refers to a sample of tissue orfluid isolated from a host, typically a human, including, but notlimited to, plasma or serum. To confirm that a factor within donorplasma is immunologically cross-reactive with one or more epitopes ofthe disclosed peptides is a straightforward matter. This can be readilydetermined using specific assays, e.g., of a single proposed epitopicsequence, or using more general screens, e.g., of a pool of randomlygenerated synthetic peptides or protein fragments. The screening assaysmay be employed to identify-either equivalent antigens or cross-reactiveantibodies. In any event, the principle is the same, i.e., based uponcompetition for binding sites between antibodies and antigens.

Any test which measures the binding of an antigen to an antibody can beused to evaluate the level of antigen or antibody in the host'sbiological sample according to the present invention. A number of othersuch tests are known and commonly used commercially.

Immunocytochemistry and immunohistochemistry are techniques that useantibodies to identify antigens on the surface of cells in solution, oron tissue sections, respectively. Immunocytochemistry is used toquantitate individual cell populations according to surface markers.Immunohistochemistry is used to localize particular cell populations orantigens. These techniques are also used for the identification ofautoantibodies, using tissues or cells that contain the presumedautoantigen as substrate. The antibodies are usually identified usingenzyme-conjugated antibodies to the original antibody, followed by achromogen, which deposits an insoluble colored end product on the cellor tissue.

Another common method of evaluation is a radioimmunoassay, in whichradiolabeled reagents are used to detect the antigen or antibody.Antibody can be detected using plates sensitized with antigen. The testantibody is applied and detected by the addition of a radiolabeledligand specific for that antibody. The amount of ligand bound to theplate is proportional to the amount of test antibody. This test can bereversed to test for antigen. Variations of radioimmunoassays arecompetition RIA, direct binding RIA, capture RIA, sandwich RIA, andimmunoradiometric assay (RMA).

Enzyme linked immunoabsorbent assays (ELISA) are a widely used group oftechniques for detecting antigen and antibodies. The principles areanalogous to those of radioimmunoassays except that an enzyme isconjugated to the detection system rather than a radioactive molecule.Typical enzymes used are peroxidase, alkaline phosphatase and2-galactosidase. These can be used to generate colored reaction productsfrom colorless substrates. Color density is proportional to the amountof reactant under investigation. These assays are more convenient thanRIA, but less sensitive.

The Western blotting (immunoblotting) method is used to characterizeunknown proteins. Components of the biological sample are separated bygel electrophoresis. SDS gels separate according to molecular weight andIEF gels separate the samples according to charge characteristics. Theseparated proteins are transferred to membranes (blotted) and identifiedby immunocytochemistry.

Less often used but suitable methods of evaluation include the Farrassay (in which radiolabeled ligands bind to and detect specificantibody in solution which are precipitated and quantified), precipitinreactions (in which antibodies and antigens crosslink into largelattices to form insoluble immune complexes; only works if antigen andantibody are present in sufficient amounts, at near equivalence, andwhen there are enough epitopes available to form a lattice);nephelometry (measures immune complexes formed in solution by theirability to scatter light); immunodiffusion (detects antigens andantibodies in agar gels); counter-current electrophoresis (similar toimmunodiffusion, except that an electric current is used to drive theantibody and antigen together; useful for low concentrations of antigenor antibody); single radial immunodiffusion (SRID)(quantitates antigensby allowing them to diffuse outward from a well into an antibodycontaining gel; technique can be reversed by diffusing unknown antibodysolutions into an antigen-containing well); rocket electrophoresis(similar to SRID, except that the test antigen is moved into the gel byan electric field); and immunofluorescence (similar to immunochemistry,except that it used fluorescence rather than enzyme conjugates). Theantibody used to contact the sample of body fluid is preferablyimmobilized onto a solid substrate. The antibody can be immobilizedusing a variety of means, as described in Antibodies: A LaboratoryManual, cited supra. Suitable solid substrates include those having amembrane or coating supported by or attached to sticks, synthetic glass,agarose beads, cups, flat packs, or other solid supports. Other solidsubstrates include cell culture plates, ELISA plates, tubes, andpolymeric membranes.

Means for labeling antibodies with detectable agents are also describedin Antibodies: A Laboratory Manual. The amount of antigen in the hostbiological sample can be determined by any means associated with theselected assay. For example, the selected immunoassay can be carried outwith known increasing amounts of antigen to produce a standard curve orcolor chart, and then the amount of test antigen can be determined bycomparing the result of the test to the standard curve or chart thatcorrelates the amount of antigen-antibody complex with known amounts ofantigen. The amount of antigen determined to be present in the hostbiological sample can be used to evaluate the patient's condition in anumber of ways. First, the level of antigen can be compared to apopulation norm based on statistical data. Second, the level of antigencan be considered in light of the patient's own history of antigenlevel.

The kit can optionally contain a lysing agent that lyses cells presentin the sample of body fluid. Suitable lysing agents include surfactantssuch as Tween-80, Nonidet P40, and Triton X-100. Preferably, the lysingagent is immobilized onto the solid support along with the antibody.

The kit can also contain a buffer solution for washing the substratebetween steps. The buffer solution is typically a physiological solutionsuch as a phosphate buffer, physiological saline, citrate buffer, orTris buffer.

The kit can optionally include different concentrations of a preformedantigen to calibrate the assay. The kit can additionally contain avisual or numeric representation of amounts of antigen in a calibratedstandard assay for reference purposes. For example, if an assay is usedthat produces a colored product, a sheet can be included that provides adepiction of increasing intensities associated with differing amounts ofantigen.

The kit can optionally include two antibodies in the detection system.The first antibody which is present in small amounts is specific for theantigen being assayed for. The second antibody provided in higheramounts is used to detect the first antibody. For example, a rabbitantibody can be used to detect the LOOH/amine antigen, and then ananti-rabbit IgG antibody can be used to detect the bound rabbitantibody. Goat antibodies and anti-antibodies are also commonly used.

As one nonlimiting example, a kit for the detection of the lipidperoxidation state of a patient is provided that includes a rabbitantibody specific for desired antibody, anti-rabbit IgG antibody insufficient amounts to detect the bound first antibody, an enzymeconjugated to the second antibody and a substrate for the enzyme whichchanges color on exposure to the enzyme.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Preparation of Prototype Four Component MSCRAMM Vaccine

A series of recombinant proteins, representing domains from thecollagen, Fn, and Fbg-binding MSCRAMMs (FIG. 1), were overexpressed inE. coli and affinity purified by metal chelating chromatography aspreviously described (see, e.g., Job et al., Biochemistry. 33(20):6086-6092, 1994; Patti et al., J. Biol. Chem. 270, 12005-12011,1995; McDevitt et al., Mol. Micro. 11 (2):237-248, 1994; Ni Eidhin etal., Infect. Immun. Submitted, 1998). Used were the following: aminoacids contained in the recombinant collagen-binding MSCRAMM expressedfrom CNA (M55, such as disclosed in co-pending U.S. patent applicationSer. No. 08/856,253, incorporated herein by reference); amino acidscontained in the recombinant fibrinogen-binding MSCRAMM-expressed fromclfA (Region A, such as disclosed in U.S. patent application Ser. No.08/293,728, incorporated herein by reference); amino acids contained inthe recombinant fibrinogen-binding MSCRAMM expressed from clfB (RegionA, such as disclosed in U.S. application Ser. No. 09/200,650,incorporated herein by reference); and amino acids contained in therecombinant fibronectin-binding MSCRAMM (DUD4, such as those disclosedin co-pending U.S. application Ser. No. 09/010,317, incorporated hereinby reference). The recombinant FN-binding MSCRAMM protein DUD4 wastreated with formalin (5% formalin overnight, 4° C.) prior to combiningit with the M55, Region A from ClfA and Region A from ClfB.

Example 2 Example of Growing E. coli Strains for Production ofRecombinant Proteins

Overnight cultures of E. coli JM101 or TOP 3 cells (Stratagene)harboring the recombinant plasmids were diluted 1:50 in 1 L of LuriaBroth (Gibco BRL) containing 50 mg/mL ampicillin. E. coli cells weregrown until the culture reached an OD₆₀₀ of 0.5-0.8. Expression of therecombinant proteins was induced by adding IPTG to a final concentrationof 0.2 mM. After a three hour induction period, cells were collected bycentrifugation, resuspended in 15 mL of Buffer A (5 mM imidazole, 0.5 MNaCl, 20 mM Tris-HCl, pH 7.9) and lysed by passage through a Frenchpress twice at 20,000 lb./in². Cell debris was removed by centrifugationat 50,000×g for 10 min and the supernatant was passed through a 0.45 μMfilter.

Example 3 Purification of HIS₆ Containing Recombinant Proteins Expressedfrom pQE-30 (Qiagen®; Qiagen Inc., Chatsworth, Calif.) or PV-4 BasedRecombinant Plasmids

The recombinant proteins were purified by immobilized metal chelatechromatography, using a column of iminodiacetic acid/Sepharose® 6B FastFlow (Sigma, St. Louis, Mo.) charged with Ni²⁺; (Porath et al. 1975;Hochuli et al. 1988). The HIS₆ tagged proteins were purified byimmobilized metal chelate affinity chromatography. More specifically, acolumn containing iminodiacetic acid Sepharose® 6B FF, connected to aFPLC® system (Pharmacia), was charged with 150 mM Ni⁺⁺ and equilibratedwith buffer A (5 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). Afterequilibration, the bacterial supernatant was applied to the column andthe column was washed with 10 bed volumes of buffer A. Subsequently, thecolumn was eluted with buffer B (200 mM imidazole, 0.5 M NaCl, 20 mMTris, pH 7.9). The eluate was monitored for protein by the absorbance at280 nm and peak fractions were analyzed by SDS-PAGE. Endotoxin wasremoved from the purified recombinant proteins by detergent extractionwith 1% Triton X-114 followed by metal chelate affinity chromatographyand passage through a polymyxcin B-sepharose column. The level ofendotoxin was quantitated using a chromogenic Limulus Amebocyte Lysate(BioWhittaker, Walkersville, Md.) assay.

Example 4 Immunization of Animals with Four Component MSCRAMMVaccine—MSCRAMM IV

Rhesus Monkeys:

100 μg of M55 (1 EU/mg), ClfA (2.5 EU/mg), ClfB (<1.0 EU/mg), and DUD4(<10 EU/mg) were mixed together to form the MSCRAMM IV vaccine. Thecocktail was mixed with TiterMax™ Gold (CytRX, Norcross, Ga.) in a 1:1ratio. Two female rhesus monkeys, ID#495Z & 664U (^(˜)9.4 kg), werevaccinated intramuscularly (IM) in the hind quadricep with 200 μl of thevaccine. Twenty-eight days later the two monkeys were boosted IM with200 μl of the same vaccine formulation. Two additional female monkeys,ID#215W & 203U (^(˜)8.0 kg), were immunized with the MSCRAMM IV that wascompounded in a 1:1 ratio with aluminum hydroxide (2% Alhydrogel;Superfos, Denmark). Twenty-eight days later the two monkeys were boostedIM with 200 μl of the same vaccine formulation.

The clinical regimen followed is described below: Day 0 15 mlpre-immunization plasma sample, complete blood chemistry Day 1 VaccinateIM hind quadricep with 0.2 ml MSCRAMM IV (100 μg), injection site exam,temperature recorded Day 7 Liver panel, temperature recorded, injectionsite exam Day 14 15 ml plasma sample Day 21 15 ml plasma sample Day 28Complete blood chemistry, temperature recorded 15 ml plasma sample,boost with IM injection of 0.2 ml MSCRAMM IV (100 μg) Day 30 Liverpanel, temperature recorded, injection site exam Day 35 Liver panel,temperature recorded, injection site, 15 ml plasma sample Day 42 15 mlplasma sample Day 49 15 ml plasma sample Day 106 15 ml plasma sample

All 4 animals seroconverted following the initial immunization. Antibodylevels >3 times above background could be detected by ELISA 106 daysafter the primary vaccination. The four animals received another boosterimmunization in the 21^(st) week of the study. Each animal was given abooster of four subcutaneous injections of 125 μl of the vaccine for atotal booster of 600 μl of the vaccine. Antibody levels at least 3 timesabove background, and as much as 15 times above background, could bedetected by ELISA 189 days after the primary vaccination. See FIG. 2. Noadverse injection site reactions were detected by direct observation byveterinarians. In addition, liver enzyme profiles, CBC, and hematologyprofiles were within the normal range for rhesus monkeys.

Example 5 Analysis of Plasma Samples from the Vaccinated Monkeys wereAnalyzed by ELISA

Immulon-2 microtiter plates (Dynex Technologies, Chantilly, Va.) werecoated overnight at 4° C. with 10 μg/ml (50 μl) of the collagen bindingMSCRAMM (M55), fibrinogen binding MSCRAMM (clfA; pCF44), fibrinogenbinding MSCRAMM (ClfB; Region A), and the fibronectin binding MSCRAMM(DUD4). Fifty microliters of the diluted plasma samples were added tothe MSCRAMM coated wells and incubated for 1 hr at room temperature.Wash buffer consisting of PBS containing 0.05% vol/vol Tween-20, ablocking solution of 1% wt/vol BSA, 0.05% Tween-20 in PBS, and antibodydilution buffer consisting of PBS containing 0.1% BSA, 0.05% Tween-20.Incubation with primary and secondary antibodies was for 60 min at 25°C. The secondary antibody was alkaline phosphatase-conjugated goatanti-monkey immunoglobulin G, (Rockland, Gilbertsville, Pa.), diluted3500-fold in antibody dilution buffer. ELISA plates were developed for30 min at 37° C. with 1 mg/ml p-nitrophenyl phosphate (Sigma) in 1 Mdiethanolamine, 0.5 mM MgCl₂, pH 9.8, and quantified at 405 nm on aPerkin Elmer HTS 7000 Bio-Assay reader. Each plasma sample was diluted100-fold in phosphate buffered saline, containing 0.05% Tween 20, 0.1%BSA, pH 7.4. ELISA data are shown in FIG. 2.

Example 6 Inhibition Assays

Methicillin resistant S. aureus strain 601 (Smeltzer, M. S., Gene.196:249-159, 1997) was cultured under constant rotation for 15 h at 37°C. in BHI broth. A 1:100 dilution of the overnight culture was made intoBHI and the bacteria were grown at 37° C. until mid exponential phase.The bacteria were harvested by centrifugation, washed three times insterile PBS, pH 7.4, and then resuspended in a carbonate buffer (50 mMNaHCO₃, pH 8.5). The bacteria were mixed with 1 mg/ml FITC (Sigma;F-7250) in 50 mM NaHCO₃, pH 8.5 and incubated end-over-end in the darkfor 1 hr at 25° C. The FITC labeling reaction was stopped bycentrifugation of the bacterial cells and removing the supernatantcontaining the unreacted FITC. The labeled bacteria were washed threetimes in PBS to remove unincorporated FITC, resuspended in PBS, adjustedto ^(˜)1×10⁸ cfu/ml and stored at −20° C. in PBS, pH 7.4.

Example 7 Purification of IgG from Immunized Monkeys

IgG was purified from the monkey plasma by affinity chromatography onPROSEP®-A high capacity resin (Bioprocessing Inc., Princeton, N.J.).Briefly, the plasma was thawed and passed through 0.45μ filter. Theplasma was applied to a benchtop column containing PROSEP®-A highcapacity resin. The unbound material was removed by washing the columnextensively with PBS. The IgG was eluted from the column with 0.1 Msodium citrate, pH 3.0. The pH of eluted IgG was immediately neutralizedto pH 6.8-7.4 by the addition of 1M Tris, pH 9.0. The IgG was thendialyzed into PBS, pH 7.4, concentrated and filter sterilized. Theconcentration of the purified IgG was determined by absorbance at 280nm.

Example 8 Competitive Inhibition ELISA

Costar 96 well black plates were coated overnight at 4° C. or at roomtemperature for 2 hr with a 10 μg/ml solution of matrix componentsconsisting of bovine collagen, human fibrinogen, and bovine fibronectinin PBS, pH 7.4. The matrix protein coated plates were washed three timeswith PBS, 0.05% Tween 20 and then blocked with PBS, 1% BSA. The blockedplates were washed three times with PBS, 0.05% Tween 20. A 500 μlaliquot of FITC-labeled S. aureus cells were mixed with an increasingamount of purified monkey IgG in PBS, 0.05% Tween 20, 0.1% BSA. Thelabeled cells and IgG were mixed on an end-over-end shaker for 1 hr at25° C. Fifty μl of the labeled cells/IgG mixture was added to each wellon the microtiter plate and incubated at 25° C. on a rocker platform.The wells were washed three times with PBS, 0.05% Tween 20. The amountof bacteria bound to the immobilized matrix proteins was determined on aPerkin Elmer HTS 7000 Bio-Assay reader with the excitation filter set at485 nm and the emission filter set at 535 nm. Data are shown in FIGS.3-5.

Example 9 Animal Model of Sepsis

Using a mouse model of sepsis (Bremell, T. A., et al., Infect. Immun. 62(7):2976-2985, 1992) we have demonstrated that passive immunization withIgG purified from rhesus monkeys immunized with the MSCRAMM IV canprotect mice against sepsis induced death. Naive male NMRI mice 5-8weeks old were passively immunized i.p. on day −1 with 20 mg of eitherpurified IgG from rhesus monkeys immunized with MSCRAMM IV (n=12), orIgG from non-immunized rhesus monkeys (n=13). On day 0, the mice werechallenged i.v. with 2.4×10⁷ CFU/mouse S. aureus strain LS-1. Mortalityand weight change was monitored over the next 3 days. Three days afterthe inoculation 3/13 mice (13%) were dead in the control group, comparedto 0/12 mice (0%) in the control group. Mortality in control group atday 13 was 53.8% (7/13) compared to only 16.2% (2/12) for the MSCRAMM IVpassively immunized group. The control mice exhibited a significantdecrease in their body weight compared to MSCRAMM IV IgG passivelyimmunized mice (28.0±2.5% vs 21.3±3.1%; p<0.01).

Example 10 ELISAs

ELISAs were performed in Immulon-II 96-well microtiter plates (DynexTechnologies, Chantilly, Va.), with wash buffer consisting of PBScontaining 0.1% vol/vol Tween-80, a blocking solution of 1% wt/vol BSA,0.1% Tween-80 in PBS, and antibody dilution buffer consisting of PBScontaining 0.05% Tween-80. Incubation with primary and secondaryantibodies was for 60 min at 25° C. The secondary antibody was alkalinephosphatase-conjugated goat anti-human immunoglobulin G, (Chemicon,Temecula, Calif.), diluted 3000-fold in antibody dilution buffer. ELISAplates were developed for 30 min at 20° C. with 1 mg/ml p-nitrophenylphosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl₂, pH 9.8 andquantified on a Molecular Dynamics ELISA plate reader equipped with a405 nm filter. Each human serum sample was diluted 100-fold in phosphatebuffered saline, containing 0.05% Tween 20, 0.1% BSA, pH 7.4. The ELISAplates were coated overnight at 4° C. with 1 μg/ml (100 μl) of thecollagen binding MSCRAMM (M55; Patti, J. M., et al., J. Biol. Chem. 270,12005-12011, 1995), fibrinogen binding MSCRAMM (clfA; pCF44; McDevitt,D., et al., Mol. Micro. 11 (2):237-248, 1994), fibrinogen bindingMSCRAMM (clfB; Region A domain; Ni Edhin, D., et al., Infect. Immun.Submitted, 1998) and the fibronectin binding MSCRAMM (DUD4; Joh, H. J.,et al., Biochemistry. 33 (20):6086-6092, 1994). One hundred microlitersof the diluted serum samples were added to the MSCRAMM coated wells andincubated for 1 hr at room temperature.

100 human plasma donor samples were analyzed using the above-describedELISA protocol. Eight donors were selected as having elevated MSCRAMMantibody titers (“MSCRAMM Selected”). Plasma units that ranged from 700ml-850 ml, from eight donors were pooled and the IgG purified byaffinity chromatography on PROSEP®-A high capacity resin (BioprocessingInc., Princeton, N.J.). Briefly, the human plasma was thawed at 4° C.for 24 hours and the units pooled into one batch. The plasma pool waspoured through cheesecloth and then filtered through 0.45μ filter. Theplasma was applied to a column of PROSEP®-A high capacity resinconnected to a preparative scale HPLC (Waters). The unbound material wasremoved by washing the column extensively with PBS. The IgG was elutedfrom the column with 0.1 M sodium citrate, pH 3.0. The pH of eluted IgGwas immediately neutralized to pH 6.8-7.4 by the addition of 1 M Tris,pH 9.0. The IgG was then dialyzed into PBS, concentrated and filtersterilized. The concentration of the purified IgG was determined byabsorbance at 280 nm.

Example 11 Animal Model of S. aureus Infection

A rabbit model of infectious endocarditis (Perlman, B. B., and L. S.Freedman, Yale J. Biol. Med. 42:394-410, 1971) was used to evaluate theprophylactic potential of the “MSCRAMM Selected” human IgG. This modelhas been used over the past decade to investigate the pathogenesis ofendocarditis and to test a variety of new antibiotics and vaccines. Inthis model, 2.5 kg rabbits underwent a transcarotid-transaortic valvularcatheterization with an indwelling polyethylene catheter. Eight rabbitswere then treated intraperitoneally with 18 ml of the “MSCRAMM Selected”human IgG (28 mg/ml; 504 mg total). Eight control rabbits received 18 mlsterile PBS and ten rabbits received 500 mg of normal human IVIG (AlphaTherapeutics Veniglobulin S; Los Angeles, Calif.) intraperitoneally.Infective endocarditis was produced 18 hours after IgG administration byan intraperitoneal injection of 10⁹ cfu S. aureus strain Reynolds. Theanimals were followed for 72 hours and blood samples were obtained at12-hour intervals. After 72 hours, the animals were euthanized and thekidneys and valvular vegetations aseptically removed. The tissue sampleswere processed for quantitative culture. Rabbits were consideredpositive for endocarditis if bacteria were recovered from thevegetations, irrespective of the bacterial density. It should be notedthat the lowest level of bacterial detection in this model is =2 log₁₀cfu/g tissue. The number of organisms recovered from the tissue sites(kidney and valvular vegetations) were statistically compared using atwo-tailed Student's t-test. P values lower than 0.05 for individualcomparisons are considered significant. The results are shown in table2. TABLE 2 MEAN FREQUENCY VEGETATION MEAN RENAL ANIMAL FREQUENCY OF OFRENAL DENSITIES DENSITIES GROUP ENDOCARDITIS SEEDING Log₁₀ cfu/g ± SDLog₁₀ cfu/g ± SD A 7/8 8/8 6.07 ± 3.33 4.48 ± 1.32 B 1/8 0/8 2.25 ±0.62* 2.00 ± O.OO{circumflex over ( )} C 10/10 10/10 7.42 ± 3.45 6.17 ±2.08Group A = PBSGroup B = MSCRAMM selected human IgGGroup C = Normal IVIG (Alpha Therapeutics Veniglobulin S)*p = 0.05 (vs. group A){circumflex over ( )}p = 0.05 (vs. group A),p = 0.001 (vs. group C)

Example 12 Tests Regarding ClfA and CNA Selected Human IVIG

A. Prophylactic Administration of ClfA and CNA Donor Selected SA-IVIGIVIG

The objective of the studies described here was to determine if passiveimmunization with donor selected IVIG products prepared from human donorplasma containing high titers of antibodies against microbial surfacecomponents recognizing adhesive matrix molecule (MSCRAMM) proteinsexpressed by Staphylococcus aureus (S. aureus) can prevent mortalitycaused by an antibiotic resistant S. aureus clinical isolate in a murinesepticemia model.

SA-IVIG MS502, S. aureus Immunoglobulin Intravenous.

Human IgG was purified using 15 units of plasma collected from 7 donorsthat were determined to possess elevated levels of IgG (>5 fold increasein titer compared to normal IVIG) in an ELISA assay specific for the Adomain of the ClfA MSCRAMM protein expressed by S. aureus. Plasma wasobtained from Serologicals, Inc. (Clarkston, Ga.) and the IgG waspurified by Cangene Corp. as a sterile-filtered solution in 10% maltose,0.03% polysorbate 80. The material was reported by Cangene to contain47.55 mg/ml IgG by radial immunodiffusion. This material was stored at4° C., as directed by the manufacturer. On the day of administration,MS502 was diluted to 40 mg/ml with 1×D-PBS in preparation for an IPinjection of 0.5 ml.

SA-IVIG MS503, S. aureus Immunoglobulin Intravenous.

Human IgG was purified using 12 units of plasma collected from 5 donorsthat were determined to possess elevated levels of IgG (>5 fold increasein titer compared to normal IVIG) in an ELISA assay specific for the Adomain of the CNA MSCRAMM expressed by S. aureus. Plasma was obtainedfrom Serologicals, Inc. (Clarkston, Ga.) and the IgG was purified byCangene Corp. as a sterile-filtered solution in 10% maltose, 0.03%polysorbate 80. The material was reported by Cangene to contain 45.41mg/ml IgG by radial immunodiffusion. This material was stored at 4° C.,as directed by the manufacturer. On the day of administration, MS503 wasdiluted to 40 mg/ml with 1×D-PBS in preparation for an IP injection of0.5 ml.

Control, Human Immune Globulin Intravenous, Polygam® (Baxter IVIG).

A sterile freeze-dried preparation of IgG was manufactured from largepools of human plasma by Baxter Healthcare Corp. The material wasreconstituted in sterile water for injection according to themanufacturer's directions (Baxter Healthcare Corp.). The 5% solutioncontains 50 mg/ml total protein, 45 mg/ml IgG, 8.5 mg/ml NaCl, 3 mg/mlhuman albumin, 22.5 mg/ml glycine, 20 mg/ml glucose, 2 mg/mlpolyethylene glycol, 1 μg/ml tri(n-butyl) phosphate, 1 μg/ml octoxynol 9and 100 μg/ml polysorbate 80. Prior to injection the stock was dilutedin sterile distilled water to a final concentration of 40 mg/ml IgG inpreparation for an IP injection of 0.5 ml.

S. aureus

S. aureus strain 601 (Smeltzer, et. al., 1996) was obtained from Dr. M.S. Smeltzer, University of Arkansas for Medical Sciences. This S. aureusstrain was isolated from an intensive care unit. The isolate iscephalothin, ciprofloxacin, clindamycin, erythromycin, oxacillin(methicillin), penicillin-G and trimethoprin-sulfamethoxazole resistant.Strain 601 S. aureus cells taken from a frozen glycerol stock, wereinoculated into a 10 ml BHI broth culture and grown over night at 37° C.Cells from the overnight culture were diluted 1:100 in BHI broth andgrown at 37° C. for approximately 3 hours until the absorbance at 600 nmreached 1.8-2.0 OD units. The bacteria were pelleted by centrifugationand resuspended in ⅕ volume of freezing medium (1×D-PBS, pH 7.4; 10%DMSO; 5% BSA). A small aliquot of the stock was plated on blood agardishes at 10⁻², 10⁻⁴ and 10⁻⁶ dilutions and cultured over night todetermine the CFU concentration of the preparation. The bacterialpreparation was stored at −86° C. until used. On the day of injection,the frozen bacterial stock was thawed and pelleted by centrifugation.The bacteria were washed once in D-PBS and resuspended to theappropriate concentration in D-PBS for IV injection. A portion of thebacterial suspension was plated on blood agar dishes at 10⁻², 10⁻⁴ and10⁻⁶ dilutions and cultured over night to determine the CFUconcentration of the final injected preparation.

Animal Sex, Species, Number, Age and Source

56 female mice (5-6 weeks of age) were purchased from Taconic QualityLaboratory Animals and Services for Research (Germantown, N.Y.). Animalswere allowed to acclimate for at least 14 days prior to initiation oftreatment. Upon arrival, the mice were examined, group housed (5/cage)in polycarbonate shoe box cages with absorbent bedding. All mice wereplaced on a 12 hour light-dark cycle under the required husbandrystandards found in the NIH Guide for the Care and Use of LaboratoryAnimals.

Identification and Randomization

All animals were uniquely identified using tail tattoos prior to dosing.Prior to initiation of treatment, the animals were individually weighedand their health was evaluated. Mice were randomized and assigned totreatment groups using stratified body weights.

Experimental Design

On Day −1, animals were treated with a single 0.5 ml IP injection ofMS502, MS503 or Baxter IVIG. On Day 0, 2.2×10⁸ CFU S. aureus wereadministered by a single IV injection (0.1 ml) to all animals via thetail vein.

DATA

Mice were pre-treated by intraperitoneal (IP) injection with 20 mg ofeither Baxter's normal IVIG product or 20 mg SA-IVIG MS502 and 20 mgSA-IVIG MS503. MS502 was an immunoglobulin G (IgG) preparation purifiedfrom donor plasma containing elevated titers of antibodies recognizingthe A domain of clumping factor (ClfA), a S. aureus fibrinogen bindingMSCRAMM protein. Likewise MS503 was a high titer preparation selectedfor recognition of the A domain of CNA, the collagen binding S. aureusMSCRAMM. The total IgG concentrations of the two MSCRAMM preparationsand the standard Baxter's normal product is provided in Table 3, below.As shown below, the high-titer MS502 sample had a total ClfA content of2.29 Units/mg as opposed to only 0.2 Units/mg in the normal sample. Thehigh-titer MS503 sample had a total CNA content of 1.06 Units/mg asopposed to only 0.2 Units/mg in the normal sample. 24 hours after IgGadministration, the mice were challenged with a single intravenous (IV)injection of a methicillin-resistant strain of S. aureus (Strain 601).The mice were followed for 5 days at which point all remaining mice weresacrificed. Significant differences in the relative survival timesbetween treatment groups were detected. Sixty-three percent (12/19) ofthe mice that received MS502 (p=0.003 vs. control; Mantel-Cox survivalanalysis) survived the bacterial challenge. Sixty-eight percent (13/19)of the mice that received MS503 (p=0.0008 vs. control; Mantel-Coxsurvival analysis) survived the bacterial challenge. Only 22% (4/18) ofthe mice treated with normal human IVIG survived the entire studyperiod. These results clearly indicate that prophylactic administrationof ClfA and CNA donor selected SA-IVIG IVIG provides a significant levelof protection against lethal infection as compared to a commerciallyavailable normal human IVIG product. TABLE 3 Total IgG Selection onConcentration ClfA CNA Product MSCRAMM (mg/ml) (Units/mg) (Units/mg)Normal Donor Unselected 45.00 0.2 0.2 Pool MS502 ClfA 47.55 2.29 NTMS503 CAN 45.41 NT 1.06NT = not testedBaxter Gammagard ® IgIV represents a normal unselected IgIVB. Therapeutic Applications of ClfA-Selected Human SA-IVIG

SA-IVIG MS502, S. aureus Immunoglobulin Intravenous.

Human IgG was purified using 15 units of plasma collected from 7 donorsthat were determined to possess elevated levels of IgG (>5 fold increasein titer compared to normal IVIG) in an ELISA assay specific for the Adomain of the ClfA MSCRAMM protein expressed by S. aureus. Plasma wasobtained from Serologicals, Inc. (Clarkston, Ga.) and the IgG waspurified by Cangene Corp. as a sterile-filtered solution in 10% maltose,0.03% polysorbate 80. The material was reported by Cangene to contain47.55 mg/ml IgG by radial immunodiffusion. This material was stored at4° C., as directed by the manufacturer. On the day of administration,MS502 was diluted to 40 mg/ml with 1×D-PBS in preparation for an IPinjection of 0.5 ml.

S. aureus

S. aureus strain 601 (Smeltzer, et. al., 1996) was obtained from Dr. M.S. Smeltzer, University of Arkansas for Medical Sciences. This S. aureusstrain was isolated from an intensive care unit. The isolate iscephalothin, ciprofloxacin, clindamycin, erythromycin, oxacillin(methicillin), penicillin-G and trimethoprin-sulfamethoxazole resistant.Strain 601 S. aureus cells taken from a frozen glycerol stock, wereinoculated into a 10 ml BHI broth culture and grown over night at 37° C.Cells from the overnight culture were diluted 1:100 in BHI broth andgrown at 37° C. for approximately 3 hours until the absorbance at 600 nmreached 1.8-2.0 OD units. The bacteria were pelleted by centrifugationand resuspended in ⅕ volume of freezing medium (1×D-PBS, pH 7.4; 10%DMSO; 5% BSA). A small aliquot of the stock was plated on blood agardishes at 10⁻², 10⁻⁴ and 10⁻⁶ dilutions and cultured over night todetermine the CFU concentration of the preparation. The bacterialpreparation was stored at −86° C. until used. On the day of injection,the frozen bacterial stock was thawed and pelleted by centrifugation.The bacteria were washed once in D-PBS and resuspended to theappropriate concentration in D-PBS for IV injection. A portion of thebacterial suspension was plated on blood agar dishes at 10⁻², 10⁻⁴ and10⁻⁶ dilutions and cultured over night to determine the CFUconcentration of the final injected preparation.

Animal Sex, Species, Number, Age and Source

Female mice (5-6 weeks of age) were purchased from Taconic QualityLaboratory Animals and Services for Research (Germantown, N.Y.). Animalswere allowed to acclimate for at least 5 days prior to initiation oftreatment. Upon arrival, the mice were examined, group housed (5/cage)in polycarbonate shoe box cages with absorbent bedding. All mice wereplaced on a 12 hour light-dark cycle under the required husbandrystandards found in the NIH Guide for the Care and Use of LaboratoryAnimals.

Identification and Randomization

All animals were uniquely identified using tail tattoos prior to dosing.Prior to initiation of treatment, the animals were individually weighedand their health was evaluated. Mice were randomized and assigned totreatment groups using stratified body weights.

Experimental Design

On Day −1, animals were treated with a single 0.5 ml IP injection ofMS502 IVIG. On Day 0, 5.6×10⁷ CFU S. aureus 601 was administered by asingle IV injection (0.1 ml) to all animals via the tail vein. Inaddition, a group of mice received MS502 IVIG 3 hours after the IVbacterial challenge. Control mice were left untreated.

DATA

Mice were treated by intraperitoneal (IP) injection with 20 mg ofSA-IVIG MS502 either 18 or prior or 3 hours after an IV challenge withS. aureus 601. MS502 was an immunoglobulin G (IgG) preparation purifiedfrom donor plasma containing elevated titers of antibodies recognizingthe A domain of clumping factor (ClfA), a S. aureus fibrinogen bindingMSCRAMM protein. The mice were followed for 5 days at which point allremaining mice were sacrificed. Ninety-three percent of the mice thatreceived MS502 SA-IVIG 18 hours prior to S. aureus challenge survived.Similarly, 93% of the mice that received MS502 SA-IVIG 3 hours postbacterial challenge survived. In contrast, only 76% of the control micesurvived the bacterial challenge. These results clearly indicate thattherapeutic administration of ClfA donor selected human SA-IVIG providesa significant and effective treatment of staphylococcal infection ascompared to a commercially available normal human IVIG product.

1. A method of obtaining a human immunoglobulin composition having ahigher antibody titer to a staphylococcal clumping factor A (ClfA)adhesin than that found in pooled intravenous immunoglobulin obtainedfrom unselected human donors comprising obtaining blood or plasmasamples from human donors, identifying those blood or plasma samplesfrom high-titer donors having the presence of an antibody titer to ClfAin an amount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors, recovering blood orplasma from the identified high-titer donors, and treating the donorblood or plasma to obtain a human immunoglobulin composition in apurified state that has an antibody titer to ClfA in an amount which ishigher than that found in intravenous immunoglobulin obtained fromunselected donors.
 2. The method according to claim 1 wherein donors areidentified which have an antibody titer to ClfA in an amount which is2-fold or greater than that found in pooled intravenous immunoglobulinobtained from unselected donors.
 3. The method according to claim 1wherein donors having a high titer to ClfA are determined by identifyingthose samples having a high titer of antibodies to the A domain of ClfA.4. The method according to claim 1 further comprising identifying thosesamples also having an antibody titer to a second staphylococcal adhesinwhich is higher than that found in pooled intravenous immunoglobulinobtained from unselected donors.
 5. The method according to claim 4wherein the second staphylococcal adhesin is an Sdr protein.
 6. Themethod according to claim 5 wherein donors having a high titer to theSdr protein are determined by identifying those samples having a hightiter of antibodies to the A domain of the Sdr protein.
 7. The methodaccording to claim 5 wherein the Sdr protein is selected from the groupconsisting of SdrF, SdrG, and SdrH.
 8. A human immunoglobulincomposition obtained by the method of claim
 1. 9. A method of obtaininga human immunoglobulin composition having a higher antibody titer to astaphylococcal ClfA adhesin than that found in pooled intravenousimmunoglobulin obtained from unselected human donors comprisingadministering ClfA to a host donor in an amount sufficient so as toinduce an antibody titer to ClfA in an amount which is higher than thatfound in pooled intravenous immunoglobulin obtained from unselecteddonors, recovering blood or plasma from the host donor, and treating thedonor blood or plasma to obtain a human immunoglobulin composition in apurified state that has an antibody titer to ClfA which is higher thanthat found in pooled intravenous immunoglobulin obtained from unselecteddonors.
 10. The method according to claim 9 wherein the host donor isinduced to have an antibody titer to ClfA in an amount which is higherthan that found in pooled intravenous immunoglobulin obtained fromunselected donors by administering the A domain of ClfA to the hostdonor an amount sufficient so as to induce an antibody titer to ClfA inan amount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors.
 11. The method accordingto claim 9 wherein immunoglobulin is obtained that has an antibody titerto ClfA in an amount which is 2-fold or greater than that found inpooled intravenous immunoglobulin obtained from unselected donors. 12.The method according to claim 9 further comprising administering asecond staphylococcal adhesin to a host donor in an amount sufficient soas to induce an antibody titer to the second adhesin in an amount whichis higher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors.
 13. The method according to claim 12 wherein thesecond staphylococcal adhesin is an Sdr protein.
 14. The methodaccording to claim 13 wherein the host donor is induced to have anantibody titer to the Sdr protein in an amount which is higher than thatfound in pooled intravenous immunoglobulin obtained from unselecteddonors by administering the A domain of the Sdr protein an amountsufficient so as to induce an antibody titer to the Sdr protein in anamount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors.
 15. The method accordingto claim 13 wherein the Sdr protein is selected from the groupconsisting of SdrF, SdrG, and SdrH.
 16. A human immunoglobulincomposition obtained by the method of claim
 9. 17. A method of obtaininga human immunoglobulin composition having a higher than normal antibodytiter to a staphylococcal Sdr protein comprising obtaining blood orplasma samples from donors, identifying those blood or plasma samplesfrom high-titer donors having the presence of an antibody titer to astaphylococcal Sdr protein in an amount which is higher than that foundin pooled intravenous immunoglobulin obtained from unselected donors,recovering blood or plasma from the identified high-titer donors, andtreating the donor blood or plasma to obtain a human immunoglobulincomposition in a purified state that has an antibody titer to astaphylococcal Sdr protein in an amount which is higher than that foundin intravenous immunoglobulin obtained from unselected donors.
 18. Themethod according to claim 17 wherein donors are identified which have anantibody titer to a staphylococcal Sdr protein in an amount which is2-fold or greater than that found in pooled intravenous immunoglobulinobtained from unselected donors.
 19. The method according to claim 18wherein donors having a high titer to a staphylococcal Sdr protein aredetermined by identifying those samples having a high titer ofantibodies to the A domain of a staphylococcal Sdr protein.
 20. Themethod according to claim 18 further comprising identifying thosesamples also having an antibody titer to a second adhesin which ishigher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors.
 21. The method according to claim 20 wherein thesecond adhesin is also a staphylococcal Sdr protein.
 22. The methodaccording to claim 21 wherein donors having a high titer to the secondstaphylococcal Sdr protein are determined by identifying those sampleshaving a high titer of antibodies to the A domain of the secondstaphylococcal Sdr protein.
 23. A human immunoglobulin compositionobtained by the method of claim
 18. 24. A method of obtaining a humanimmunoglobulin composition having a higher than normal antibody titer toa staphylococcal Sdr protein comprising administering a staphylococcalSdr protein to a host donor in an amount sufficient so as to induce anantibody titer to a staphylococcal Sdr protein in an amount which ishigher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors, recovering blood or plasma from the host donor,and treating the donor blood or plasma to obtain a human immunoglobulincomposition in a purified state that has an antibody titer to astaphylococcal Sdr protein which is higher than that found in pooledintravenous immunoglobulin obtained from unselected donors.
 25. Themethod according to claim 24 wherein the host donor is induced to havean antibody titer to a staphylococcal Sdr protein in an amount which ishigher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors by administering the A domain of a staphylococcalSdr protein to the host donor in an amount sufficient so as to induce anantibody titer to a staphylococcal Sdr protein in an amount which ishigher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors.
 26. The method according to claim 24 whereinimmunoglobulin is obtained that has an antibody titer to astaphylococcal Sdr protein in an amount which is 2-fold or greater thanthat found in pooled intravenous immunoglobulin obtained from unselecteddonors.
 27. The method according to claim 24 further comprisingadministering a second adhesin to a host donor in an amount sufficientso as to induce an antibody titer to the second adhesin in an amountwhich is higher than that found in pooled intravenous immunoglobulinobtained from unselected donors.
 28. The method according to claim 27wherein the second adhesin is a second staphylococcal Sdr protein. 29.The method according to claim 28 wherein the host donor is induced tohave an antibody titer to the second staphylococcal Sdr protein in anamount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors by administering the Adomain of the second staphylococcal Sdr protein an amount sufficient soas to induce an antibody titer to the second staphylococcal Sdr proteinin an amount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors.
 30. A humanimmunoglobulin composition obtained by the method of claim
 24. 31. Amethod of obtaining an immunoglobulin composition having a higherantibody titer to a staphylococcal clumping factor A (ClfA) adhesin thanthat found in pooled intravenous immunoglobulin obtained from unselectedhuman donors comprising obtaining blood or plasma samples from humandonors, and: (a) identifying those blood or plasma samples fromhigh-titer donors having the presence of an antibody titer to ClfA in anamount which is higher than that found in pooled intravenousimmunoglobulin obtained from unselected donors and identifying thosesamples also having an antibody titer to a second staphylococcal adhesinselected from the group consisting of a fibronectin binding protein, acollagen binding protein, a fibrinogen binding protein, an elastinbinding protein and an MHC-II analogous protein in an amount which ishigher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors. (b) recovering blood or plasma from theidentified high-titer donors, and (c) treating the recovered blood orplasma to obtain immunoglobulin in a purified state that has an antibodytiter to ClfA in an amount which is higher than that found in pooledintravenous immunoglobulin obtained from unselected donors and anantibody titer to the second staphylococcal adhesin in an amount whichis higher than that found in pooled intravenous immunoglobulin obtainedfrom unselected donors;
 32. The method according to claim 31 wherein thesecond staphylococcal adhesin is selected from the group consisting ofproteins fibronectin binding protein A (FnBP-A), fibronectin bindingprotein B (FnBP-B), clumping factor protein B (ClfB), SdrC, SdrD, SdrE,SdrF, SdrG, SdrH, CNA, and EbpS.
 33. The method of claim 31 whereindonors having a high titer to the staphylococcal Sdr protein aredetermined by identifying those samples having a high titer ofantibodies to the A domain of the staphylococcal Sdr protein.